Stabilized vancomycin formulations

ABSTRACT

In one aspect, the invention provides a stabilized lipid-based glycopeptide antibiotic composition and a process for producing the same. In another aspect, the invention provides methods for treating a bacterial pulmonary infection by administering to a subject in need thereof a therapeutically effective amount of the stabilized lipid-based glycopeptide antibiotic composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 61/731,363, filed Nov. 29, 2012, which is incorporated herein byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Vancomycin is a branched tricyclic glycosylated non ribosomal peptideantibiotic produced by the fermentation of the Actinobacteria speciesAmycolaopsis orientalis, and is believed to act by inhibiting propercell wall synthesis in Gram-positive bacteria. Additionally, it isbelieved that vancomycin alters cell membrane permeability and RNAsynthesis. Accordingly, vancomycin is generally used in the prophylaxisand treatment of infections caused by Gram-positive bacteria that areunresponsive to other types of antibiotics.

Vancomycin has been reported as a treatment of last resort forinfections that are resistant to other first line antibiotics. This isbecause vancomycin is given intravenously for most indications.Additionally, vancomycin presents toxicity concerns, and semi-syntheticpenicillins have been developed and used preferentially over vancomycin.Nevertheless, the use of vancomycin has increased particularly with thespread of multiple-resistant Staphylococcus aureus (MRSA).

Methods for treating pulmonary disorders using liposomal vancomycinformulations are described in U.S. Publication Nos. US 2009-0105126 andUS 2009-0104257, and U.S. Provisional Application Nos. 61/103,725 and60/981,990, all of which are hereby incorporated by reference in theirentireties. There is a need in the art for cost-effective vancomycinformulations that degrade at a slower rate and therefore exhibitimproved stability. The present invention addresses these and otherneeds.

SUMMARY OF THE INVENTION

In one aspect, a stabilized lipid-based glycopeptide antibioticcomposition is provided. In one embodiment, the composition comprises alipid component, a glycopeptide antibiotic, and an amino acid or aderivative thereof. In a further embodiment, the amino acid orderivative thereof binds to the glycopeptide antibiotic and forms astabilized glycopeptide antibiotic-amino acid complex. In even a furtherembodiment, the stabilized glycopeptide antibiotic-amino acid complex isentrapped by, or complexed with, the lipid component. In one embodiment,the antibiotic is vancomycin. In another embodiment, the antibiotic isteicoplanin, telavancin, oritavancin, decaplanin or dalbavancin. In oneembodiment, the lipid component is a mixture of two or three lipids.

In one embodiment, a pharmaceutical composition comprising a lipid-basedglycopeptide antibiotic is provided. In a further embodiment, thecomposition comprises a lipid component, a glycopeptide antibiotic, andan amino acid or derivative thereof. In a further embodiment, the aminoacid or the derivative thereof stabilizes the glycopeptide antibiotic.In a yet further embodiment, the antibiotic and amino acid are entrappedby, or complexed with, the lipid. In one embodiment, the antibiotic isvancomycin, teicoplanin, telavancin, oritavancin, decaplanin ordalbavancin. In a further embodiment, the antibiotic is vancomycin.

In one embodiment, a stabilized lipid-based vancomycin formulation isprovided that produces product degradants at a rate less than about0.05% by weight per week at 4° C. In another embodiment, a stabilizedlipid-based vancomycin composition is provided, and the compositionproduces product degradants at a rate less than about 0.03% by weightper week at 4° C. In a further embodiment, a stabilized lipid-basedvancomycin composition is provided that produces product degradants at arate less than about 0.02% by weight per week at 4° C. In yet a furtherembodiment, a stabilized lipid-based vancomycin composition is providedthat produces product degradants at a rate less than about 0.01% byweight per week at 4° C. In one embodiment, the product degradants arecrystalline degradation products.

In one embodiment, a stabilized lipid-based vancomycin formulation isprovided that produces product degradants at a rate less than about 0.5%by weight per week at room temperature (RT). In another embodiment, astabilized lipid-based vancomycin composition is provided that producesproduct degradants at a rate less than about 0.4% by weight per week atRT. In a further embodiment, a stabilized lipid-based vancomycincomposition is provided where the composition produces productdegradants at a rate less than about 0.2% by weight per week at RT. Inone embodiment, the product degradants are crystalline degradationproducts.

In one embodiment, the stabilized lipid-based glycopeptide antibioticcomposition comprising a lipid component, a glycopeptide antibiotic, andan amino acid or derivative thereof is at least about 44% more stable,or at least about 55% more stable, or at least about 66% more stable, orat least about 77% more stable, or at least about 88% more stable than alipid-based glycopeptide antibiotic composition that does not comprisean amino acid or derivative thereof.

In another embodiment, the present invention relates to a stabilizedlipid-based glycopeptide antibiotic comprising a lipid component, aglycopeptide antibiotic, and an amino acid or a derivative thereof,wherein the lipid component comprises a phospholipid. In a furtherembodiment, the phospholipid is phosphatidylcholine (PC),phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine(PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), eggphosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), eggphosphatidylinositol (EPI), egg phosphatidylserine (EPS),phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soyphosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soyphosphatidylserine (SPS), soy phosphatidylinositol (SPI), soyphosphatidylethanolamine (SPE), soy phosphatidic acid (SPA),hydrogenated egg phosphatidylcholine (HEPC), hydrogenated eggphosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol(HEPI), hydrogenated egg phosphatidylserine (REPS), hydrogenatedphosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA),hydrogenated soy phosphatidylcholine (HSPC), hydrogenated soyphosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS),hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soyphosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid(HSPA), dipalmitoylphosphatidylcholine (DPPC),dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol(DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), di stearoylphosphatidylglycerol(DSPG), dioleoylphosphatidylcholine (DOPC),dioleylphosphatidylethanolamine (DOPE),palmitoylstearoylphosphatidyl-choline (PSPC),palmitoylstearolphosphatidylglycerol (PSPG),mono-oleoyl-phosphatidylethanolamine (MOPE), tocopherol, tocopherolhemisuccinate, cholesterol sulfate, cholesteryl hemisuccinate,cholesterol derivatives, ammonium salts of fatty acids, ammonium saltsof phospholipids, ammonium salts of glycerides, myristylamine,palmitylamine, laurylamine, stearylamine, dilauroyl ethylphosphocholine(DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoylethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP),N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP), di stearoylphosphatidylglycerol (DSPG),dimyristoylphosphatidylacid (DMPA), dipalmitoylphosphatidylacid (DPPA),di stearoylphosphatidylacid (DSPA), dimyristoylphosphatidylinositol(DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol (DSPI), dimyristoylphosphatidylserine(DMPS), dipalmitoylphosphatidylserine (DPPS),distearoylphosphatidylserine (DSPS), or mixtures thereof. In oneembodiment, the lipid component comprises DPPC. In another embodiment,the lipid component comprises DPPG. In another embodiment, the lipidcomponent comprises DPPC and DPPG. In one embodiment, the lipidcomponent comprises one lipid, two lipids or three lipids. In yetanother embodiment, the lipid component consists of one lipid, twolipids or three lipids.

In another embodiment, a stabilized lipid-based glycopeptide antibioticcomprising a sterol, a glycopeptide antibiotic, and an amino acid or aderivative thereof is provided. In one embodiment, the sterol ischolesterol. In another embodiment, the composition further comprisesDPPC.

In another embodiment, the present invention comprises a stabilizedlipid-based glycopeptide antibiotic comprising a lipid componentcomprising a phospholipid and a sterol, a glycopeptide antibiotic, andan amino acid or a derivative thereof. In one embodiment, thephospholipid and sterol are dipalmitoylphosphatidylcholine (DPPC) andcholesterol, respectively. In another embodiment, the phospholipid andsterol are dipalmitoylphosphatidylglycerol (DPPG) and cholesterol. Inanother embodiment, the phospholipid and sterol comprise DPPC, DPPG andcholesterol.

In one embodiment, the present invention comprises a stabilizedlipid-based glycopeptide antibiotic comprising a lipid component, aglycopeptide antibiotic, and an amino acid or a derivative thereof,wherein the amino acid or derivative thereof is conjugated to theglycopeptide antibiotic to form a stabilized glycopeptideantibiotic-amino acid complex. In a further embodiment, the stabilizedglycopeptide antibiotic-amino acid complex is entrapped by the lipidcomponent. In a further embodiment, the lipid component is in a lipidclathrate, proliposome, micelle or liposome. In a further embodiment,the liposome has a mean particle size of about 0.05 to about 10 microns,0.05 to about 1 microns, 0.05 to about 0.5 microns, about 0.1 to about5.0 microns, about 0.1 to about 3.0 microns, about 0.1 to about 2.0microns, about 0.1 to about 1.0 microns, about 0.1 to about 0.5 microns,about 0.1 to about 0.4 microns, about 0.1 to about 0.3 microns, or about0.1 to about 0.2 microns. In another embodiment, the mean particularsize of the liposome is about 1.0 microns or less, about 0.9 microns orless, about 0.8 microns or less, about 0.7 microns or less, about 0.6microns or less, about 0.5 microns or less, about 0.4 microns or less,about 0.3 microns or less, or about 0.2 microns or less.

In one embodiment, the present invention provides a stabilizedlipid-based glycopeptide antibiotic composition comprising a lipidcomponent, vancomycin, and an amino acid or a derivative thereof. In oneembodiment, the amino acid is D-alanine. In another embodiment, theamino acid is aspartic acid. In another embodiment, the amino acidderivative is bicine. In another embodiment, the amino acid isD-glutamic acid. In another embodiment, the amino acid derivative isglycylglycine (GLY-GLY). In yet another embodiment, the amino acidderivative is iminodiacetic acid (IDAA). In one embodiment, thevancomycin is conjugated to the amino acid or derivative thereof.

In one embodiment, a stabilized lipid-glycopeptide antibiotic comprisinga lipid component, a glycopeptide antibiotic, and an amino acid orderivative thereof is provided, wherein the molar ratio of theglycopeptide antibiotic to the amino acid derivative thereof is fromabout 1:1 to about 1:4. In a further embodiment, the molar ratio of theglycopeptide antibiotic to the amino acid or derivative thereof is fromabout 1:1 to about 1:2. In a further embodiment, the molar ratio of theglycopeptide antibiotic to the amino acid or derivative thereof is about1:1. In another embodiment, the molar ratio of the glycopeptideantibiotic to the amino acid or derivative thereof is about 1:2.

In another embodiment, the present invention relates to a stabilizedlipid-glycopeptide antibiotic composition comprising a lipid component,a glycopeptide antibiotic, and an amino acid or derivative thereof,wherein the weight ratio of the total lipid component to theglycopeptide antibiotic is from about 0.1:1 to about 5:1. In a furtherembodiment, the weight ratio of the lipid component to the glycopeptideantibiotic is about 3:1 or less. In a further embodiment, the weightratio of the lipid component to the glycopeptide antibiotic is about 1:1or less. In another embodiment, the weight ratio of the lipid componentto the glycopeptide antibiotic is less than 1:1.

In another aspect of the invention, a method for preparing a stabilizedlipid-based glycopeptide antibiotic composition is provided. In someembodiments, the glycopeptide antibiotic is vancomycin. In a furtherembodiment, the method comprises mixing a first stream of a lipidsolution containing a lipid in a solvent with a second stream of anaqueous solution comprising a glycopeptide antibiotic (e.g., vancomycin)and an amino acid or a derivative thereof. In one embodiment, the mixingof the two streams comprises infusing, in an in-line fashion, the firststream of the lipid solution with the second stream of the aqueoussolution containing the glycopeptide antibiotic and amino acid orderivative thereof. In a further embodiment, the glycopeptide antibioticand amino acid or derivative thereof is present as a conjugated complex.In a further embodiment, the glycopeptide antibiotic-amino acid complexis entrapped by the lipid when mixed in an in-line fashion. In a furtherembodiment, the solvent is ethanol. In another embodiment, the firststream of lipid solution is provided at a first flow rate and the secondstream of aqueous solution is provided at a second flow rate. In afurther embodiment, the first flow rate is about 1 L/min and the secondflow rate is about 1.5 L/min.

In one embodiment, the second stream of the aqueous solution comprisesthe amino acid D-alanine. In another embodiment, the amino acid in theaqueous solution is aspartic acid. In another embodiment, the amino acidderivative in the aqueous solution is bicine. In another embodiment, theamino acid in the aqueous solution is D-glutamic acid. In anotherembodiment, the amino acid derivative in the aqueous solution isgycylglycine (GLY-GLY). In another embodiment, the amino acid derivativein the aqueous solution is iminodacetic acid (IDAA).

In yet another aspect of the invention, a method for treating abacterial pulmonary infection with a stabilized glycopeptide antibioticcomposition is provided. In one embodiment, the method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of an amino acid stabilized lipid-based glycopeptide antibioticcomposition. In a further embodiment, the glycopeptide antibiotic isvancomycin. In another embodiment, the bacterial pulmonary infection iscaused by a Gram-positive bacteria selected from the group consisting ofStaphylococcus, Streptococcus, Enterococcus, Bacillus, Corynebacterium,Nocardia, Clostridium, and Listeria. In a further embodiment, theGram-positive bacteria are selected from the group consisting ofMethicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli,Klebsiella, Enterobacter, Serratia, Haemophilus, Yersinia pesos,Burkholderia pseudomallei, Burkholderia cepacia, Burkholderia gladioli,Burkholderia multivorans, Burkholderia vietnamiensis, Mycobacteriumtuberculosis, Mycobacterium avium complex (MAC)(Mycobacterium avium andMycobacterium intracellulare), Mycobacterium kansasii, Mycobacteriumxenopi, Mycobacterium marinum, Mycobacterium mucogenicum, Mycobacgeriumgordonae, Mycobacterium ulcerans, and Mycobacterium fortuitum complex(including, but not limited to, Mycrobacterium fortuitum, Mycrobacteriumperegrinum, Mycrobacterium chelonae, Mycrobacterium abscessus, andMycrobacterium mucogenicum.

In one embodiment, a method for treating a pulmonary disease with astabilized glycopeptide antibiotic composition is provided. In oneembodiment, the pulmonary disease is cystic fibrosis, bronchiectasis,pneumonia, or chronic obstructive pulmonary disease (COPD). In anotherembodiment, a method for treating osteomyelitis; endocarditis;bronchitis; hepatitis; myocarditis; nephritis; bacteremia; a skin orconnective tissue infection including, but not limited to, folliculitis,cellulitis, furuncules, or pymyositis; or a wound or surgical siteinfection with a stabilized glycopeptide antibiotic composition isprovided.

In one embodiment, a composition comprising a stabilized lipid-basedglycopeptide antibiotic for use in the therapy of cystic fibrosis,bronchiectasis, pneumonia, or chronic obstructive pulmonary disease(COPD) is provided. In another embodiment, a composition comprising astabilized lipid-based glycopeptide antibiotic for use in the therapy ofosteomyelitis; endocarditis; bronchitis; hepatitis; myocarditis;nephritis; bacteremia; a skin or connective tissue infection including,but not limited to, folliculitis, cellulitis, furuncules, or pymyositis;or a wound or surgical site infection is provided. In one embodiment,the composition for use in the therapy comprises a lipid component, aglycopeptide antibiotic, and an amino acid or a derivative thereof. In afurther embodiment, the amino acid or derivative thereof binds to theglycopeptide antibiotic and forms a stabilized glycopeptideantibiotic-amino acid complex. In even a further embodiment, thestabilized glycopeptide antibiotic-amino acid complex is entrapped by,or complexed with, the lipid component. In one embodiment, theantibiotic is vancomycin. In another embodiment, the antibiotic isteicoplanin, telavancin, oritavancin, decaplanin or dalbavancin.

In one embodiment, a composition comprising a stabilized lipid-basedglycopeptide antibiotic for use as a medicament in the treatment ofcystic fibrosis, bronchiectasis, pneumonia, or chronic obstructivepulmonary disease (COPD) is provided. In another embodiment, acomposition comprising a stabilized lipid-based glycopeptide antibioticfor use as a medicament in the treatment of osteomyelitis; endocarditis;bronchitis; hepatitis; myocarditis; nephritis; bacteremia; a skin orconnective tissue infection including, but not limited to, folliculitis,cellulitis, furuncules, or pymyositis; or a wound or surgical siteinfection is provided. In one embodiment, the composition for use as amedicament comprises a lipid component, a glycopeptide antibiotic, andan amino acid or a derivative thereof. In a further embodiment, theamino acid or derivative thereof binds to the glycopeptide antibioticand forms a stabilized glycopeptide antibiotic-amino acid complex. Ineven a further embodiment, the stabilized glycopeptide antibiotic-aminoacid complex is entrapped by, or complexed with, the lipid component. Inone embodiment, the antibiotic is vancomycin. In another embodiment, theantibiotic is teicoplanin, telavancin, oritavancin, decaplanin ordalbavancin.

In one embodiment, a composition comprising a stabilized lipid-basedglycopeptide antibiotic for use in the manufacture of a medicament forcystic fibrosis, bronchiectasis, pneumonia, or chronic obstructivepulmonary disease (COPD) is provided. In another embodiment, acomposition comprising a stabilized lipid-based glycopeptide antibioticfor use in the manufacture of a medicament for osteomyelitis;endocarditis; bronchitis; hepatitis; myocarditis; nephritis; bacteremia;a skin or connective tissue infection including, but not limited to,folliculitis, cellulitis, furuncules, or pymyositis; or a wound orsurgical site infection is provided. In one embodiment, the compositionfor use in the manufacture of a medicament comprises a lipid component,a glycopeptide antibiotic, and an amino acid or a derivative thereof. Ina further embodiment, the amino acid or derivative thereof binds to theglycopeptide antibiotic and forms a stabilized glycopeptideantibiotic-amino acid complex. In even a further embodiment, thestabilized glycopeptide antibiotic-amino acid complex is entrapped by,or complexed with, the lipid component. In one embodiment, theantibiotic is vancomycin. In another embodiment, the antibiotic isteicoplanin, telavancin, oritavancin, decaplanin or dalbavancin.

In another embodiment, the therapeutically effective amount of astabilized lipid-based glycopeptide antibiotic composition is an amountgreater than a minimum inhibitory concentration (MIC) for the bacterialpulmonary infection. In another embodiment, the therapeuticallyeffective amount of a stabilized lipid-based glycopeptide antibioticcomposition is a dose of about 50 to 1000 mg/day, about 100 to 500mg/day, or about 250 to 500 mg/day. In a further embodiment, the dose isabout 100 mg/day. In other embodiments, the dose is about 200 mg, about300 mg, about 400 mg, or about 500 mg per day. In another embodiment,the composition is administered 1 time to 4 times a day. In a furtherembodiment, the composition is administered once a day, twice a day,three times a day or four times a day. In another embodiment, thecomposition is administered in a daily treatment cycle for a period oftime, or is administered in a cycle of every other day, every third day,every fourth day, every firth day, every 6^(th) day or once a week for aperiod of time, the period of time being from one week to severalmonths, for example, 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, or 6 months.

In another embodiment, the lipid based glycopeptide antibioticcomposition is administered by inhalation as a nebulized spray, powder,or aerosol. In a further embodiment, the stabilized lipid basedglycopeptide composition is administered via a nebulizer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of vancomycin.

FIG. 2 shows the structural changes during vancomycin degradation toCrystalline Degradation Product-I (CDP-I).

FIG. 3 shows the general structure of four types of natural glycopeptideantibiotics.

FIG. 4 is a typical chromatogram of vancomycin using a ZIC-HILIC column.CDP-I-M, CDP-I-m, and Vancomycin peaks are shown at retention times of13, 5, 4.9, and 23.9, respectively.

FIG. 5 is a graph of vancomycin degradation over time at pH of 5, 5.5,6, or 6.5, at 4° C., and at a concentration of 200 mg/mL (top panel) or20 mg/mL (bottom panel).

FIG. 6 is a graph of vancomycin degradation over time in NaOH(diamonds); NaOH with EtOH (squares); Tris-base (triangle symbols);Tri-Ethanol Amine (TEOA; Xs); or Ethanol Amine (EOA; asterisks) at 4° C.and at pH 6 (top panel) or pH 6.5 (bottom panel).

FIG. 7 is a bar graph of the rate of degradation of vancomycin at 4° C.in the presence of the indicated organic buffers at the indicated pH.

FIG. 8 is a graph of the degradation of vancomycin over time at pH of 5,5.5, 6, or 6.5, at room temperature (RT), and at a concentration of 200mg/mL (top panel) or 20 mg/mL (bottom panel).

FIG. 9 is a graph of the degradation of vancomycin over time in thepresence of NaOH (diamonds); NaOH with EtOH (squares); Tris-base(triangles); Tri-Ethanol Amine (TEOA; Xs); or Ethanol Amine (EOA;asterisks), at room temperature (RT), and at pH 6 (top panel) or pH 6.5(bottom panel).

FIG. 10 is a graph of vancomycin degradation rates at room temperature(RT) in the presence of the indicated organic buffers and at theindicated pH.

FIG. 11 is a graph of vancomycin degradation over time (top panel) andvancomycin degradation rates (bottom panel) in vancomycin compositionswith the indicated amino acid or derivative thereof at 4° C. Amino acidswere added on a mole per mole basis unless otherwise stated. In the toppanel, the vancomycin compositions comprise: control (NaOH alone, pH 5,dark diamonds); bALA (dark squares); ALA (dark triangles); GABA, pH5.5(light circles); GLY (dark line with X symbols); D-ALA (dark line withasterisk symbols); 3-ABA (dark circles); GLU (dark line with plussymbols); G-GLU (dark line with no symbols); ASP (medium line with nosymbols); D-ASP (light diamonds); bicine (light squares); tricine (lighttriangles); sarcosine (light line with Xs); IDAA (light line withasterisks); GLY-GLY (light line with plus symbols); GLU, pH5 (2:1 ratioof GLU-vancomycin, pH5; lighter line with no symbols); and GLU, pH5.5(2:1 ratio of GLU-vancomycin, pH5.5; lightest line with no symbols).

FIG. 12 is a graph of vancomycin degradation over time (top panel) andvancomycin degradation rates (bottom panel) in vancomycin compositionswith the indicated amino acid or derivative thereof at RT. Amino acidswere added on a mole per mole base unless otherwise stated. In the toppanel, the vancomycin compositions comprise: control (NaOH alone, pH 5,light line with plus symbol); bALA (dark diamonds); ALA (dark squares);GABA, pH5.5 (dark triangles); GLY (light circles); D-ALA (dark line withX symbols); 3-ABA (dark line with asterisk symbols); GLUt (darkcircles); G-GLUt (dark line with plus symbols); ASP (dark line with nosymbols); D-ASP (medium line with no symbols); bicine (light diamonds);tricine (light squares); sarcosine (light triangles); IDAA (light linewith asterisk symbols); GLY-GLY (light line with plus symbols); GLU pH5(2:1 ratio of GLU-vancomycin, pH5; light line with X symbols); and GLUpH5.5 (2:1 ratio of GLU-vancomycin, pH5.5; light line with asterisksymbols).

FIG. 13 shows an Arrhenius plot for vancomycin degradation rates basedon Table 14. Vancomycin in NaOH at pH 5.5 (diamonds); Bicine-vancomycinin a 2:1 ratio (squares); GLY-GLY-vancomycin in a 1:1 ratio (triangles);GLY-GLY-vancomycin in a 1.5:1 ratio (Xs); GLY-GLY-vancomycin in a 2:1ratio (asterisks); and GLU-vancomycin in a 2:1 ratio (circles).

FIG. 14 shows a liposomal vancomycin infusion diagram (three streaminfusion process).

FIG. 15 is a graph of the stability over time of liposomalvancomycin-GLU compositions at 4° C. The batches of liposomal vancomycincompositions tested are those shown in Table 15 (i.e., diamondscorrespond to L-VGLU0330, comprising GLU and DPPC/cholesterol; squarescorrespond to L-VDGLU0405, comprising D-GLU and DPPC/DPPG/cholesterol;and triangles correspond to LPG-VGLU0408, comprising GLU andDPPC/cholesterol).

FIG. 16 is a graph of the stability over time of liposomalvancomycin-GLU compositions at RT. The batches of liposomal vancomycincompositions tested are those shown in Table 15 (i.e., diamondscorrespond to L-VGLU0330, comprising GLU and DPPC/cholesterol; squarescorrespond to L-VDGLU0405, comprising D-GLU and DPPC/DPPG/cholesterol;and triangles correspond to LPG-VGLU0408, comprising GLU andDPPC/cholesterol).

FIG. 17 is a graph of stability over time of liposomal vancomycin-GLUcompositions at 4° C. The batches of liposomal vancomycin compositionstested are those shown in Table 15 (i.e., diamonds correspond toL-VGLU0330, comprising GLU and DPPC/cholesterol; and squares correspondto L-VDGLU0405, comprising D-GLU and DPPC/DPPG/cholesterol).

FIG. 18 is a graph of stability over time of liposomal vancomycin-GLUcompositions at RT. The batches of liposomal vancomycin compositionstested are those shown in Table 15 (i.e., diamonds correspond toL-VGLU0330, comprising GLU and DPPC/cholesterol; and squares correspondto L-VDGLU0405, comprising D-GLU and DPPC/DPPG/cholesterol).

DETAILED DESCRIPTION OF THE INVENTION

The abbreviations used herein for amino acids are those abbreviationswhich are conventionally used: A=Ala=Alanine; R=Arg=Arginine;N=Asn=Asparagine; D=Asp=Aspartic acid; C=Cys=Cysteine; Q=Gln=Glutamine;E=Glu=Gutamic acid; G=Gly=Glycine; H=His=Histidine; I=Ile=lsoleucine;L=Leu=Leucine; K=Lys=Lysine; M=Met=Methionine; F=Phe=Phenylalanine;P=Pro=Proline; S=Ser=Serine; T=Thr=Threonine; W=Trp=Tryptophan;Y=Tyr=Tyrosine; V=Val=Valine. The amino acids in the compositionsprovided herein are L- or D-amino acids. In one embodiment, a syntheticamino acid is used in the compositions provided herein. In oneembodiment, the amino acid increases the half-life, efficacy and/orbioavailability of the glycopeptide antibiotic in the composition. In afurther embodiment, the glycopeptide antibiotic is vancomycin.

The term “amino acid derivative” as used herein refers to a moietyhaving both an amine functional group, either as NH₂, NHR, or NR₂, and acarboxylic acid functional group, either as NH₂, NHR, or NR₂, and acarboxylic acid functional group. The term “amino acids” encompassesboth natural and unnatural amino acids, and can refer to alpha-aminoacids, beta-amino acids, or gamma amino acids. Unless specifiedotherwise, an amino acid structure referred to herein can be anypossible stereoisomer, e.g., the D or L enantiomer. In some embodiments,the amino acid derivatives are short peptides, including dipeptides andtripeptides. Exemplary amino acids and amino acid derivatives suitablefor the invention include alanine (ALA), D-alanine (D-ALA),alanine-alanine (ALA-ALA), beta-alanine (bALA), alanine-beta-alanine(ALA-bALA), 3-aminobutanoic acid (3-ABA), gamma-aminobutyric acid(GABA), glutamic acid (GLU or GLUt), D-glutamic acid (D-GLU), glycine(GLY), glycylglycine (GLY-GLY), glycine-alanine (GLY-ALA),alanine-glycine (ALA-GLY), aspartic acid (ASP), D-aspartic acid (D-ASP),lysine-alanine-alanine (LYS-ALA-ALA), L-Lysine-D-alanine-D-alanine(L-LYS-D-ALA-D-ALA), bicine, tricine, sarcosine, and iminodiacetic acid(IDAA). Amino acids and derivatives thereof can be synthesized accordingto known techniques, or can be purchased from suppliers, e.g.,Sigma-Aldrich (Milwaukee, Wis.).

The term “administering” includes in vivo administration, as well asadministration directly to tissue ex vivo. Generally, compositions maybe administered systemically either orally, buccally, parenterally,topically, by inhalation or insufflation (i.e., through the mouth orthrough the nose), or rectally in dosage unit formulations containingconventional nontoxic pharmaceutically acceptable carriers, adjuvants,and vehicles as desired, or may be locally administered by means suchas, but not limited to, injection, implantation, grafting, topicalapplication, or parenterally.

In one embodiment, the composition of the invention is administered viainhalation. In a further embodiment, the composition of the invention isadministered via nebulization, vaporization, aerosolization, or drypowder inhalation. A “nebulizer” or an “aerosol generator” in oneembodiment, is used to administer the compositions of the presentinvention to a patient in need thereof. A “nebulizer” or an “aerosolgenerator” is a device that converts a liquid into an aerosol of a sizethat can be inhaled into the respiratory tract. Pneumonic, ultrasonic,electronic nebulizers, e.g., passive electronic mesh nebulizers, activeelectronic mesh nebulizers and vibrating mesh nebulizers are amenablefor use with the invention if the particular nebulizer emits an aerosolwith the required properties, and at the required output rate.

The process of pneumatically converting a bulk liquid into smalldroplets is called atomization. The operation of a pneumatic nebulizerrequires a pressurized gas supply as the driving force for liquidatomization. Ultrasonic nebulizers use electricity introduced by apiezoelectric element in the liquid reservoir to convert a liquid intorespirable droplets. Various types of nebulizers are described inRespiratory Care, Vol. 45, No. 6, pp. 609-622 (2000), the disclosure ofwhich is incorporated herein by reference in its entirety. The terms“nebulizer” and “aerosol generator” are used interchangeably throughoutthe specification. “Inhalation device”, “inhalation system” and“atomizer” are also used in the literature interchangeably with theterms “nebulizer” and “aerosol generator.”

“Mass median aerodynamic diameter” or “MMAD” is normalized regarding theaerodynamic separation of aqua aerosol droplets and is determined byimpactor measurements, e.g., the Anderson Cascade Impactor (ACI) or theNext Generation Impactor (NGI). The gas flow rate, in one embodiment, is28 Liter per minute by the Anderson Cascade Impactor (ACI) and 15 literper minute by the Next Generation Impactor (NGI). “Geometric standarddeviation” or “GSD” is a measure of the spread of an aerodynamicparticle size distribution.

In one embodiment, the MMAD of the aerosol of the pharmaceuticalcomposition is less than about 4.9 μm, less than about 4.5 μm, less thanabout 4.3 μm, less than about 4.2 μm, less than about 4.1 μm, less thanabout 4.0 μm or less than about 3.5 μm, as measured by the ACI at a gasflow rate of about 28 L/minute, or by the Next Generation Impactor (NGI)at a gas flow rate of about 15 L/minute.

In one embodiment, the MMAD of the aerosol of the pharmaceuticalcomposition is about 1.0 μm to about 4.2 μm, about 3.2 μm to about 4.2μm, about 3.4 μm to about 4.0 μm, about 3.5 μm to about 4.0 μm or about3.5 μm to about 4.2 μm, as measured by the ACI. In one embodiment, theMMAD of the aerosol of the pharmaceutical composition is about 2.0 μm toabout 4.9 μm, about 4.4 μm to about 4.9 μm, about 4.5 μm to about 4.9μm, or about 4.6 μm to about 4.9 μm, as measured by the NGI.

“Fine particle fraction” or “FPF”, as used herein, refers to thefraction of the aerosol having a particle size less than 5μm indiameter, as measured by cascade impaction. FPF is usually expressed asa percentage.

In one embodiment, the fine particle fraction (FPF) of the compositionpost nebulization, i.e., the aerosolized pharmaceutical composition, isabout 50%, or about 55%, or about 60%, or about 65%, or about 70%, orabout 75%, as measured by NGI or ACI. In a further embodiment, the FPFof the aerosol is greater than or equal to about 64%, as measured by theACI, greater than or equal to about 70%, as measured by the ACI, greaterthan or equal to about 51%, as measured by the NGI, or greater than orequal to about 60%, as measured by the NGI.

In one embodiment, the FPF of the aerosolized composition is greaterthan or equal to 50%, greater than or equal to 60%, greater than orequal to 70%, greater than or equal to 80%, greater than or equal to90%, greater than or equal to 95%, greater than or equal to 97.5%, orgreater than or equal to 99%, as measured by cascade impaction. In afurther embodiment, the composition comprises vancomycin and a liposome.

The terms “carrier,” “excipient,” and “vehicle” are used interchangeablyherein and refer to materials suitable for formulation andadministration of the pharmaceutically acceptable compositions describedherein. Carriers useful herein include any materials known in the artwhich are nontoxic, do not interact with other components, do not causesignificant irritation to an organism, and do not abrogate thebiological activity and properties of the compound of the composition ofthe described invention. Carriers must be of sufficiently high purityand of sufficiently low toxicity to render them suitable foradministration to the mammal being treated. The term “pharmaceuticallyacceptable salt” means those salts which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humansand lower animals without undue toxicity, irritation, allergic responseand the like and are commensurate with a reasonable benefit/risk ratio.The term “coacervation” refers to a separation into two liquid phases incolloidal systems. The phase more concentrated in the colloid component(active agent) is referred herein as the “coacervate,” and the otherphase is the equilibrium solution. Coacervate formation leads to higherinternal active agent concentrations relative to external active agentconcentrations and lower lipid to drug ratios.

The term “colloidal” refers to being of or relating to or having theproperties of a colloid, meaning aggregates of atoms or molecules in afinely divided state (submicroscopic), dispersed in a gaseous, liquid orsolid medium, and resisting sedimentation, diffusion, and filtration. Asolution of macromolecules is a simple and the most common colloidsystem. Small molecules also can form association colloids as reversibleaggregates. An association colloid is a reversible chemical combinationdue to weak chemical bonding forces wherein up to hundreds of moleculesor ions aggregate to form colloidal structures with sizes of from about1 to about 2000 nanometers or larger.

The term “effective amount” refers to the amount necessary or sufficientto realize a desired biologic effect.

The term “hydrophilic” refers to a material or substance having anaffinity for polar substances, such as water. The term “lipophilic”refers to preferring or possessing an affinity for a non-polarenvironment compared to a polar or aqueous environment.

The term “solvent” as used herein refers to a substance capable ofdissolving another substance (“a solute”) to form a solution.

“Solvent infusion” is a process that includes dissolving one or morelipids in a small, minimal, amount of a process compatible solvent toform a lipid suspension or solution and then adding the solution to anaqueous medium containing bioactive agents. Typically a processcompatible solvent is one that can be washed away in an aqueous processsuch as dialysis. The composition that is cool/warm cycled in oneembodiment, is formed by solvent infusion. In one embodiment, thesolvent is an alcohol. In a further embodiment, the alcohol is ethanol.“Ethanol infusion” is a type of solvent infusion, and is a process thatincludes dissolving one or more lipids in a small, minimal, amount ofethanol to form a lipid solution and then adding the solution to anaqueous medium containing bioactive agents. A “small” amount of solventis an amount compatible with forming liposomes or lipid complexes in theinfusion process. The term “solvent infusion” also includes an in-lineinfusion process where two streams of formulation components are mixedin-line.

The term “symptom” as used herein refers to a phenomenon that arisesfrom and accompanies a particular disease or disorder and serves as anindication of it.

The term “therapeutic effect” refers to a consequence of treatment, theresults of which are judged to be desirable and beneficial. Atherapeutic effect may include, directly or indirectly, the arrest,reduction, or elimination of a disease manifestation. A therapeuticeffect may also include, directly or indirectly, the arrest reduction orelimination of the progression of a disease or condition, or delay inthe recurrence of a disease or condition.

The term “treat” or “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease, conditionor disorder, substantially ameliorating clinical or esthetical symptomsof a condition, substantially preventing the appearance of clinical oresthetical symptoms of a disease, condition, or disorder, and protectingfrom harmful or annoying symptoms. The term “treat” or “treating” asused herein further refers to accomplishing one or more of thefollowing: (a) reducing the severity of the disorder; (b) limitingdevelopment of symptoms characteristic of the disorder(s) being treated;(c) limiting worsening of symptoms characteristic of the disorder(s)being treated; (d) limiting recurrence of the disorder(s) in patientsthat have previously had the disorder(s); and (e) limiting recurrence ofsymptoms in patients that were previously symptomatic for thedisorder(s).

The term “minimum inhibitory concentration” or “MIC” as used hereinrefers to the lowest concentration of an antimicrobial agent that willinhibit visible growth of a microorganism after overnight incubation(this period is extended for organisms such as anaerobes, which requireprolonged incubation for growth). “MIC₁₀,” “MIC₅₀,” or “MIC₉₀,” as usedherein, refer to the concentration of an antimicrobial agent that willinhibit growth of the microorganism by 10%, 50%, or 90%, respectively.When no subscript is used, MIC, it is assumed that it is the MIC₅₀ thatis being discussed. The range of antibiotic concentrations used fordetermining MICs is accepted universally to be in doubling dilutionsteps up and down from 1 mg/mL as required (Andrews, J., J. Antimicrob.Chemother., 2001, 48, (Supp1.1), 5-16, incorporated herein in itsentirety).

In one aspect of the invention, a stabilized lipid-based glycopeptideantibiotic composition is provided, comprising a lipid component, aglycopeptide antibiotic and an amino acid or a derivative thereof,wherein the amino acid or derivative thereof is conjugated to theglycopeptide antibiotic. The conjugation of the amino acid or derivativethereof to the glycopeptide antibiotic forms a stabilized glycopeptideantibiotic-amino acid complex. In one embodiment, the glycopeptideantibiotic-amino acid complex is associated with the lipid. For example,in one embodiment, the lipid is conjugated (e.g., bound) to theglycopeptide antibiotic-amino acid complex. In one embodiment, theglycopeptide antibiotic-amino acid complex is entrapped by the lipidcomponent, for example, where the lipid is in the form of a liposome.

The composition described herein, in one embodiment, comprises aliposome, proliposome, lipid colloidal dispersion, micelle, invertedmicelle, discoid structure, or a combination thereof. In a furtherembodiment, the composition comprises a liposome. The glycopeptideantibiotic and amino acid or derivative thereof, in one embodiment, iscomplexed to the liposome, or encapsulated by the liposome.

“Encapsulated” and “encapsulating” are used to refer to adsorption ofactive agents on the surface of a lipid based formulation, anassociation of active agents in the interstitial region of bilayers orbetween two monolayers, capture of active agents in the space betweentwo bilayers, or capture of active agents in the space surrounded by theinner most bilayer or monolayer.

The lipids used in the compositions of the present invention can besynthetic, semi-synthetic or naturally-occurring lipids, includingphospholipids such as phosphatidylglycerols (PGs), phosphatidic acids(PAs), phosphotidylcholines (PCs), phosphatidylinositols (PIs), andphosphatidylserines (PSs); fatty acids; ammonium salts of fatty acids;tocopherols; tocopherol derivatives; sterols; sterol derivatives; andglycerides. The fatty acids have carbon chain lengths of from 12 to 26carbon atoms, which are either saturated or unsaturated. The lipids canbe anionic, cationic, or neutral, where neutral includes both unchargedlipids and zwitterionic lipids. According to one embodiment, the lipidcomponent is substantially free of anionic lipids. According to anotherembodiment, the lipid component comprises only neutral lipids. Accordingto another embodiment, the lipid component is free of anionic lipids.

According to another embodiment, the lipid in the composition comprisesa phospholipid. Phospholipids are comprised of ester linkages of fattyacids in the 2 and 3 of glycerol positions containing chains of 12 to 26carbon atoms, and different head groups in the 1 position of glycerolthat include choline, glycerol, inositol, serine, ethanolamine, as wellas the corresponding phosphatidic acids. The chains on these fatty acidscan be saturated or unsaturated, and the phospholipid can be made up offatty acids of different chain lengths and different degrees ofunsaturation.

Almost all biologically occurring phospholipids are constructed fromcombinations of apolar and “backbone” moieties: a glycerol (or otherpolyol) moiety substituted with one or two acyl or alkyl chains or anN-acylated sphingoid base (i.e., a ceramide). Typically, the hydroxylgroup at position 3 of the glycerol is esterified to phosphoric acid,whereas the hydroxyl groups at positions 1 and 2 of the glycerol areesterified with long chain fatty acids, which provide the lipidcharacteristic of the phospholipid. One of the remaining oxygen groupsof phosphoric acid can be esterified further to a variety of organicmolecules including glycerol, choline, ethanolamine, serine, andinositol. The phosphate moiety along with the attached alcohol representthe head group of phospholipid. The fatty acid part of a phospholipid isimportant in that differences in the fatty acid part can change thecharacteristics of the phospholipid. Fatty acids can differ in thelength of their carbon chain (e.g., short, medium, or long chain) and inthe level of saturation.

In one embodiment, one or more phospholipids are present in thecomposition of the present invention. The most abundant phospholipid inplants and animals is phosphatidylcholine (also known as lecithin) andphosphatidylethanolamine, which constitute the major structural part ofmost biological membranes. In phosphatidylserine, the phosphoric acidmoiety is esterified to the hydroxyl group of the amino acid L-serine,whereas, in phosphatidylinositol, the phosphoric acid moiety isesterified to the cyclic sugar alcohol inositol. The other type ofphospholipid found in human is phosphatidylglycerol, which is a naturalcomponent of the lung surfactant. In the case of phosphatidylglycerol,the alcohol that is esterified to the phosphate moiety is glycerolinstead of phosphoric acid (Vemuri, S. and Rhodes, C., 1995,Pharmaceutica Acta Helvetiae 70: 95-111).

Examples of phospholipids that can be used in the composition of thepresent invention include, but are not limited to, phosphatidylcholine(PC), phosphatidylglycerol (PG), phosphatidylinositol (PI),phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidicacid (PA), egg phosphatidylcholine (EPC), egg phosphatidylglycerol(EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS),phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soyphosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soyphosphatidylserine (SPS), soy phosphatidylinositol (SPI), soyphosphatidylethanolamine (SPE), soy phosphatidic acid (SPA),hydrogenated egg phosphatidylcholine (HEPC), hydrogenated eggphosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol(HEPI), hydrogenated egg phosphatidylserine (REPS), hydrogenatedphosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA),hydrogenated soy phosphatidylcholine (HSPC), hydrogenated soyphosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS),hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soyphosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid(HSPA), dipalmitoylphosphatidylcholine (DPPC),dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol(DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), di stearoylphosphatidylglycerol(DSPG), dioleoylphosphatidylcholine (DOPC),dioleylphosphatidylethanolamine (DOPE),palmitoylstearoylphosphatidyl-choline (PSPC),palmitoylstearolphosphatidylglycerol (PSPG),mono-oleoyl-phosphatidylethanolamine (MOPE), tocopherol, tocopherolhemisuccinate, cholesterol sulfate, cholesteryl hemisuccinate,cholesterol derivatives, ammonium salts of fatty acids, ammonium saltsof phospholipids, ammonium salts of glycerides, myristylamine,palmitylamine, laurylamine, stearylamine, dilauroyl ethylphosphocholine(DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoylethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP),N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP), di stearoylphosphatidylglycerol (DSPG),dimyristoylphosphatidylacid (DMPA), dipalmitoylphosphatidylacid (DPPA),di stearoylphosphatidylacid (DSPA), dimyristoylphosphatidylinositol(DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol (DSPI), dimyristoylphosphatidylserine(DMPS), dipalmitoylphosphatidylserine (DPPS),distearoylphosphatidylserine (DSPS), or a mixture thereof.

In one embodiment, the composition comprises DPPC and/or DPPG.

According to another embodiment, the phospholipid used in thecomposition of the present invention is a mixed phospholipid, including,but not limited to, palmitoylstearoylphosphatidylcholine (PSPC) andpalmitoylstearoylphosphatidylglycerol (PSPG)), triacylglycerol,diacylglycerol, ceramide, sphingosine, sphingomyelin, and singleacylated phospholipids, such as mono-oleoyl-phosphatidylethanol amine(MOPE).

In one embodiment, the lipid component of the present inventioncomprises one or more sterols. In a further embodiment, the sterol ischolesterol. The sterols, of which cholesterol and its derivatives arethe most widely studied in mammalian systems, constitute a component ofmembrane lipids, along with the glycerophospholipids and sphingomyelins(Bach et al., 2003, Biochem. Biophys. Acta., 1610: 187-197). Sterollipids are subdivided primarily on the basis of biological function.There are many examples of unique sterols from plant, fungal, and marinesources that are designated as distinct subclasses of sterols. These aresubdivided on the basis of the number of carbons in the core skeleton.The C₁₈ sterols include the estrogen family, whereas the C₁₉ sterolscomprise the androgens, such as testosterone and androsterone. The C₂₁subclass, containing a two carbon side chain at the C₁₇ position,includes the progestogens as well as the glucocorticoids andmineralocorticoids. The secosterols, comprising various forms of vitaminD, are characterized by cleavage of the B ring of the core structure,hence the “seco” prefix. Additional classes within the sterols categoryare the bile acids, which in mammals are primarily derivatives ofcholan-24-oic acid synthesized from cholesterol in the liver and theirconjugates (sulfuric acid, taurine, glycine, glucuronic acid, andothers) (Fahy, E. et al., 2005, J. Lipid Res., 46:839-861). Each of thepublications referenced in this paragraph are incorporated by referenceherein in their entireties.

As provided herein, in one embodiment, the lipid component of thepresent invention comprises cholesterol. Cholesterol is found in animalmembranes and has been used in the preparation of liposomes to improvebilayer characteristics of the liposomes. The cholesterol moleculeorients itself among the phospholipid molecules with its hydroxyl groupfacing towards the water phase, the tricyclic ring sandwiched betweenthe first few carbons of the fatty acyl chains, into the hydrocarboncore of the bilayer (Vermuri, S. and Rhode, C., Pharmaceutica ActaHelvetiae, 1995, 70: 95-111). Cholesterol improves the fluidity of thebilayer membrane, reduces the permeability of water soluble moleculesthrough the membrane, and improves the stability of the bilayer membranein the presence of biological fluids such as blood/plasma. Liposomeswithout cholesterol tend to react with blood proteins, such as albumin,m-transferrin, and macroglobulin, which tend to destabilize theliposomes and reduce the utility of liposomes as drug delivery systems.

According to another embodiment, the lipid component consistsessentially of a phosphatidylcholine. According to another embodiment,the lipid component consists essentially ofdipalmitoylphosphatidylcholine (DPPC). According to another embodiment,the lipid component consists essentially ofpalmitoyloleoylphosphatidylcholine (POPC).

According to another embodiment, the lipid component consistsessentially of phosphatidylglycerol. According to another embodiment,the lipid component consists essentially of1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG).

According to one embodiment, the stabilized glycopeptide antibiotic isentrapped by or complexed with the lipid component. In one embodiment,the lipid component is in the form of a liposome. Liposomes arecompletely closed lipid bilayer membranes containing an entrappedaqueous volume. It has been reported that liposomes spontaneously formwhen phospholipids are dispersed in aqueous medium (Bangham et al.,1974, In Korn, E.D. ed., Methods in Membrane Biology, Vol. 1. PlenumPress, New York, pp. 1-68, incorporated by reference herein in itsentirety). The hydrophilic interaction of the lipid head groups withwater results in the formation of vesicles composed of simple lipidbilayers that resemble biological membranes, in the form of a sphericalshell. Liposomes can be unilamellar vesicles (possessing a singlemembrane bilayer) or multilamellar vesicles (onion-like structurescharacterized by multiple membrane bilayers, each separated from thenext by an aqueous layer), or a combination thereof. The bilayer iscomposed of two lipid monolayers having a hydrophobic “tail” region anda hydrophilic “head” region. The structure of the membrane bilayer issuch that the hydrophobic (nonpolar) “tails” of the lipid monolayersorient toward the center of the bilayer while the hydrophilic “heads”orient towards the aqueous phase.

Depending on the method of preparation, liposomes of the invention varywidely in size (e.g., 0.02-10 μm) and in the number of lamellae (i.e.,number of bilayers present within a liposome). Typically, liposomes areclassified into three categories on the basis of their size andlamellarity: small unilamellar vesicles (SUVs), small oligolamellarvesicles (OLVs), large unilamellar vesicles (LUVs), and multilamellarvesicles (MLVs). More detailed classification based on structure isshown in Table 1. (Smad, A. et al., Current Drug Delivery, 2007, 4:297-305, incorporated by reference herein in its entirety).

According to one embodiment, the pharmaceutical composition comprises aliposome or a plurality of liposomes. The glycopeptides antibiotic andamino acid (or derivative thereof) is entrapped by the liposome (orplurality of liposomes), complexed to the liposome (or plurality ofliposomes), for example, complexed to the bilayer of the liposome, or acombination of entrapped and complexed.

In one embodiment, the liposome is a unilamellar vesicle (UV). Accordingto further embodiment, the liposome is a small unilamellar vesicle(SUV), a medium unilamellar vesicle (MUV), a large unilamellar vesicle(LUV) or a giant unilamellar vesicle (GUV). According to anotherembodiment, the liposome is an oligolamellar vesicle (OV). According toanother embodiment, the plurality of liposomes comprise liposome is amultilamellar vesicle (MV) and unilamellar vesicles. According toanother embodiment, the liposome comprises a multilamellar vesicle (MV).

TABLE 1 Vesicle Types with their Size and Number of Lipid layers Numberof Vesicle Type Abbreviation Diameter Lipid Bilayer Unilamellar vesicleUV All size One ranges Small Unilamellar vesicle SUV 20-100 nm OneMedium Unilamellar vesicle MUV >100 nm One Large Unilamellar vesicleLUV >100 nm One Giant Unilamellar vesicle GUV >1 μm One Oligolamellarvesicle OLV 0.1- 1 μm Approximately 5 Multilamellar vesicle MLV >1 μm5-25 Multi vesicular vesicle MV >1 μm Multi compartmental structure

According to one embodiment, the liposome has a mean particle size ofabout 0.05 to about 10 microns, 0.05 to about 1 microns, 0.05 to about0.5 microns, about 0.1 to about 5.0 microns, about 0.1 to about 3.0microns, about 0.1 to about 2.0 microns, about 0.1 to about 1.0 microns,about 0.1 to about 0.5 microns, about 0.1 to about 0.4 microns, about0.1 to about 0.3 microns, or about 0.1 to about 0.2 microns. In anotherembodiment, the mean particular size of the liposome is about 1.0microns or less, about 0.9 microns or less, about 0.8 microns or less,about 0.7 microns or less, about 0.6 microns or less, about 0.5 micronsor less, about 0.4 microns or less, about 0.3 microns or less, or about0.2 microns or less.

According to one embodiment, the lipid component of the presentinvention is in the form of a micelle or plurality of micelles. Manysurfactants can assemble in a bulk solution into aggregates or micelles.The concentration at which surfactants begin to form micelles is knownas the “critical micelle concentration” (“CMC”). The lipid in thecompositions provided herein, in one embodiment, is a surfactant with anextremely low CMC.

According to another embodiment, the stabilized glycopeptide antibioticis entrapped by a lipid clathrate. A lipid clathrate is athree-dimensional, cage-like structure employing one or more lipidswherein the structure entraps a bioactive agent. Such clathrates areincluded in the scope of the present invention.

According to anther embodiment, the stabilized glycopeptide antibioticis entrapped by a proliposome or plurality of proliposomes. Proliposomesare formulations that can become liposomes or lipid complexes uponcoming in contact with an aqueous liquid, and include for example drypowders. Agitation or other mixing may be necessary. Such proliposomesare included in the scope of the present invention.

The tissue distribution and clearance kinetics of drug-containingliposomes are known to be affected by lipid composition and surfacecharge (Juliano and Stamp, Biochem. Biophys. Res. Commun., 1975, 63:651, incorporated by reference herein in its entirey). There are anumber of synthetic phospholipids available and utilized in thepreparation of liposomes. Gangliosides, a class of sphingolipids,sometimes are included in liposome formulations to provide a layer ofsurface charged groups, which provide longer circulating liposomes inthe blood stream. Such liposome formulations comprising sphingolipidsare included in the scope of the present invention.

The composition of the present invention includes one or moreglycopeptides antibiotics. Glycopeptide antibiotics, includingvancomycin and teicoplanin, are large, rigid molecules that inhibit alate stage in bacterial cell wall peptidoglycan synthesis. Glycopeptidesare characterized by a multi-ring peptide core containing six peptidelinkages, an unusual triphenyl ether moiety, and sugars attached atvarious sites. Over 30 antibiotics designated as belonging to theglycopeptide class have been reported. Among the glycopeptides,vancomycin and teicoplanin are used widely and are recommended fortreatment of severe infections, especially those caused bymultiple-drug-resistant Gram-positive pathogens. The glycopeptideavoparcin has been introduced as a growth promoter in animal husbandryin the past, and represents the main reservoir for the VanA type ofvancomycin resistance in enterococci. Semisynthetic derivatives ofvancomycin and teicoplanin, lipoglycopeptides, showed an extendedspectrum of activity against multi-resistant and partlyvancomycin-resistant bacteria (Reynolds P., Eur. J. Clin MicrobiolInfect Dis, 1989, 8: 943-950; Renolds, 1989; Nordmann et al., Curr.Opin. Microbiol., 2007, 10: 436-440). Each of the publicationsreferenced in this paragraph are incorporated by reference herein intheir entireties.

Glycopeptide antibiotics are active against Gram-positive organisms anda few anaerobes. The main indications for glycopeptide antibiotics areinfections caused by beta-lactamase-producing Staphylococcus aureus (forwhich beta-lactamase-resistant penicillins, cephalosporins, andcombinations of penicillins with inhibitors of beta-lactamases provedsafer alternatives), and colitis caused by Colstridium difficile. Theemergence and rapid spread of methicillin-resistant S. aureus (MRSA)strains, which were resistant not only to all beta-lactams but also tothe main antibiotic classes, renewed the interest in vancomycin andpushed teicophalnin, another natural glycopeptide, onto the market.Teicoplanin is comparable to vancomycin in terms of activity, butpresents pharmacokinetic advantages, such as prolonged half-life,allowing for a once-daily administration (van Bambeke F., Curr. Opin.Pharm., 4(5):471-478).

Prior to 1984, the glycopeptide class included few members beyondvancomycin, teicoplanin, ristocetin, and avoparcin. With theacknowledgement of the threat posed by antibiotic resistance, the classexpanded to include thousands of natural and semi-synthetic compounds.Structural studies on these compounds have clarified the biological modeof action and have served as a basis for reasonable predictionsregarding structure-activity relationships.

The structures of hundreds of natural and semisynthetic glycopeptideshave been determined. These structures are highly related and fallwithin five structural subtypes, I-V. Of the varying structuralsubtypes, type I structures contain aliphatic chains, whereas types II,III, and IV include aromatic side chains within these amino acids.Unlike types I and II, types III and IV contain an extra F-O-G ringsystem. Type IV compounds have, in addition, a long fatty-acid chainattached to the sugar moiety. Structures of type V, such ascomplestatin, chloropeptin I, and kistamincin A and B, contain thecharacteristic tryptophan moiety linked to the central amino acid. Thestructures of the subtypes are known in the art and described inNicolaou et al. (Angew. Chem. Int. Ed., 1999, 38: 2096-2152,incorporated by reference herein in its entirey), the entire contents ofwhich are incorporated herein by reference. Compounds of each of thestructural subtypes mentioned above can be used in the compositionsdescribed herein.

Biochemical studies of the mode of action of glycopeptide antibioticsindicate that these substances inhibit cell wall peptidoglycansynthesis. Treatment of intact bacteria with vancomycin atconcentrations close to the minimum inhibitory concentration (MIC)resulted in the accumulation of cytoplasmically located wall precursors(Reynolds et al., Biochimica et Biophysica Acta, 1961, 52: 403-405;Jordan. Biochemical and Biophysical Research Communications, 1961, 6:167-170), suggesting that glycopeptides interfered with a late stage inthe assembly of the peptidoglycan. Vancomycin, and other glycopeptides,cannot penetrate the cytoplasmic membrane (Perkins et al., BiochemicalJournal, 1970, 116: 83-92), and thus the critical transglycosylationreaction is the first to be inhibited (Jordan and Reynolds: Vancomycin.In: Corcoran, J. W., Hahn, F. E. (ed.): Antibiotics, volume III.Mechanism of action of antimicrobial and antitumor agents.Springer-Verlag, Berlin, 1974, 704-718.). Inhibition of this reactionresults in the accumulation of lipid intermediates in the biosyntheticpathway and of UDP-MurNAc-pentapeptide in the cytoplasm. Each of thepublications referenced in this paragraph are incorporated by referenceherein in their entireties.

Glycopeptide antibiotics tend to be unstable in solution, resulting in aloss of activity. Stability of glycopeptides can be enhanced using oneof the two peptides Ac-D-Ala-D-Ala and Di-Ac-L-Lys-D-Ala-D-Ala (Harriset al., 1985, J. Antibiot, 38(1):51-7, incorporated by reference hereinin its entirety). However, a need exists for more improvement instability of glycopeptide antibiotics, preferably in a cost-effectivemanner. Unexpectedly, the compositions of the present invention,comprising a glycopeptide antibiotic, an amino acid or derivativethereof, and a lipid component, exhibited superior stability comparedglycopeptide antibiotic compositions that are not lipid-based and/or donot comprise an amino acid or derivative thereof.

A representative number of glycopeptides that can be used in thecompositions of the present invention are provided in Table 2. Theantibiotic complexes are listed in alphabetical order along with thestructure type producing organism. These metabolites are elaborated by adiverse group of actinomycetes ranging from the more prevalentStreptomyces species to the relatively rare genera of Streptosporangiumand Saccharomonospora. The less common Actionplanes and Amycolatopsisaccount for almost half of the producing organisms (Nagarajan, R.,Glycopeotide Antibiotics, CRC Press, 1994, incorporated by referenceherein in its entirety).

TABLE 2 Glycopeptide Antibiotics and Producing Organisms Antibiotic TypeProducing Organism A477 ND Actinoplanes sp. NRRL 3884 A35512 IIIStreptomyces candidus NRRL 8156 A40926 IV Actinomadura sp. ATTC39727A41030 III Streptomyces virginiae NRRL 15156 A42867 I Nocardia sp. ATTC53492 A47934 III Streptomyces toyocaensis NRRL 15009 A80407 IIIKibdelosporangium phihppinensis NRRL 18198 or NRRL 18199 A82846 IAmycolatopsis orientalis NRRL 18100 A83850 I Amycolatopsis albus NRRL18522 A84575 I Streptosporangium carneum NRRL 18437, 18505 AB-65 NDSaccharomonospora viride T-80 FERM-P 2389 Actaplanin III Actinoplanesmissouriensis ATCC 23342 Actinoidin II Proactinomyces actinoides ArdacinIV Kibdelosporangium aridum ATCC 39323 Avoparcin II Streptomycescandidus NRRL 3218 Azureomycin ND Pseudonocardia azurea NRRL11412Chloroorienticin I Amyclolatopsis orientalis PA-45052 ChloropolysporinII Micropolyspora sp. FERM BP-538 Decaplanin I Kibdelosporangiumdeccaensis DSM 4763 N-demethyl- I Amycolatopsis orientalis NRRL 15252vancomycin Eremomycin I Actinomycetes sp. INA 238 Galacardin IIActinomycetes strain SANK 64289 FERM P-10940 Helvecardin IIPseudonocardia compacta subsp. helvetica Izupeptin ND Norcardia AM-5289FERM P-8656 Kibdelin IV Kibdelosporangium aridum ATCC 39922 LL-AM374 NDStreptomyces eburosporeus NRRL 3582 Mannopeptin ND Streptomyces platenisFS-351 MM45289 I Amycolatopsis orientalis NCIB 12531 MM47761 IAmycolatopsis orientalis NCIB 12608 MM47766 II Amycolatopsis orientalisNCBI 40011 MM55266 IV Amycolatopsis sp. NCIB 40089 MM55270 NDAmycolatopsis sp. NCIB 40086 OA-7653 I Streptomyces hygromscopicus ATCC31613 Orienticin I Nocardia orientalis FERM BP-1230 Parvodicin IVActinomadura parvosata ATCC 532463 Ristocetin III Amycolatopsisorientalis subsp. lurida NRRL 2430 Ristomycin III Proactinomycesfructfferi Synmonicin II Synnemomyces mamnoorii ATCC 53296 TeicoplaninIV Actinoplanes teichomyceticus ATCC 31121 UK-68597 III ActinoplanesATCC 53533 UK-69542 III Saccharothix aerocolonigenes UK-72051 IAmycolatopsis orientalis Vancomycin I Amycolatoposis orientalis NRRL2450

According to another embodiment, the glycopeptide antibiotic used in thecomposition of the present invention includes, but is not limited to,A477, A35512, A40926, A41030, A42867, A47934, A80407, A82846, A83850,A84575, AB-65, Actaplanin, Actinoidin, Ardacin, Avoparcin, Azureomycin,Chloroorienticin Chloropolysporin, Decaplanin, N-demethylvancomycin,Eremomycin, Galacardin, Helvecardin Izupeptin, Kibdelin, LL-AM374,Mannopeptin, MM45289, MM47761, MM47766, MM55266, MM55270, OA-7653,Orienticin, Parvodicin, Ristocetin, Ristomycin, Synmonicin, Teicoplanin,UK-68597, UK-69542, UK-72051, vancomycin, and a mixture thereof.

According to one embodiment, the glycopeptide antibiotic of the presentinvention is vancomycin. Vancomycin is a water soluble amphotericglycopeptide bactericidal antibiotic that inhibits gram-positivebacterial mucopeptide biosynthesis. It consists of a tricyclicnonribosomal heptapeptide core structure to which is attached adisaccharide unit consisting of the aminodeoxy sugar, vancosamine, andD-glucose (FIG. 1). This natural antibiotic of ˜1450 Daltons is obtainedfrom Streptomyces orientalis (also known as; Nocardia orientalis, orAmycolatopsis orientalis). Vancomycin has one carboxyl group with pKa2.18, and two amino groups: primary amine with pKa 7.75 and thesecondary amine with pKa 8.89. At sub-physiological pH vancomycin has anet positive charge.

Although vancomycin has been reported to be bactericidal, it is notnecessarily the case that the bacteria are killed. Without wishing to bebound by theory, the bacteria are instead prevented from growing by thesaturation of the available growth points of the peptidoglycan. Thenon-covalent nature of the binding of vancomycin to the important targetsites is indicated by the ease with which the inhibition of eitherbacterial growth or peptidoglycan synthesis could be reversed. Suchreversal has been accomplished by the addition to the growth orincubation medium of a suitable peptide that competed effectively withthe natural wall peptides at the growth points for the availableglycopeptide (Nieto, M. et al., Biochemical Journal, 1972, 126: 139-149,incorporated by reference herein).

Studies have shown that vancomycin binds reversibly to theL-Lys-D-Ala-D-Ala fragment of the peptidoglycan monomer. Thisreversible, noncovalent interaction inhibits transglycosidation andtranspeptidation from occurring. Inhibition of these processes leads tothe collapse of the peptidoglycan by decisively shifting its dynamicequilibrium towards de-assembly, which precipitates cell lysis andbacterial death.

The strong binding of vancomycin to L-Lys-D-Ala-D-Ala is a consequenceof five well-defined hydrogen bonds. In Gram-positive bacteria, theglycopeptide antibiotics easily diffuse through the peptidoglycan layerand reach the periplastic space where the peptidoglycan polymerizationtakes place. By grabbing onto the L-Lys-D-Ala-D-Ala tails of themonomers the antibiotic positions itself to inhibit the transglycosidasefrom joining the carbohydrate.

Due to the nature of the manufacturing process for vancomycin, the rawmaterial usually contains a number of impurities. In addition,vancomycin is not very stable in solution and degrades to severalproducts, known as product degradants. It is believed that the maindegradation product of vancomycin is crystalline degradation product one(CDP-I), and results from deamidation of an asparagine residue. Thestructural changes during vancomycin degradation to CDP-I are shown inFIG. 2. CDP-I has limited aqueous solubility and exists in two isomericforms often referred to as CDP-I-m (minor) and CDP-I-M (major). Theseare the atropisomers involving different orientations of the CIsubstituted aromatic ring of residue 2. The order of the CDP formationis thought to be: vancomycin→succinimide intermediate→CDP-I-m→CDP-I-M(Harris, C. et al., 1983, Journal of the American Chemical Society, 105(23):6915-6922, incorporated by reference herein). In equilibrium, theratio of the two forms in solution is approximately 1:2 and theequilibrium time is 24 hours at pH 6.5 at 25° C. CDP-I can be convertedfurther into crystalline hydrochloride CDP-II by incubating with 0.6NHCl (Marshall, 1965, J. Med Chem, 8:18-22, incorporated by referenceherein in its entirety). CDP-II seems to not form in measurablequantities under normal conditions.

According to one embodiment, the glycopeptide antibiotic of the presentinvention is oritavancin (LY333328). Oritavancin is obtained byreductive alkylation with 4′ chloro-biphenylcarboxaldehyde of thenatural glycopeptide chloroeremomycin, which differs from vancomycin bythe addition of a 4-epi-vancosamine sugar and the replacement of thevancosamine by a 4-epivancosamine (Cooper, R. et al., J Antibiot (Tokyo)1996, 49:575-581, incorporated by reference herein in its entirety). Thestructure of oritavancin is shown in FIG. 3. Although oritavancinpresents a general spectrum of activity comparable to that ofvancomycin, it offers considerable advantages in terms of intrinsicactivity (especially against streptococci), and remains insensitive tothe resistance mechanisms developed by staphylococci and enterococci.Because the binding affinity of vancomycin and oritavancin to freeD-Ala-D-Ala and D-Ala-D-Lac are of the same order of magnitude, thedifference in their activity has been attributed to the cooperativeinteractions that can occur between the drug and both types ofprecursors in situ. The previous study suggested that the effect iscaused possibly by a much stronger ability to dimerize and the anchoringin the cytosolic membrane of the chlorobiphenyl side chain (Allen, etal., FEMS Microbiol Rev, 2003, 26:511-532, incorporated by referenceherein).

The efficacy of oritavancin has been demonstrated in animal models ofmeningitis caused by pneumococci susceptible or resistant to β-lactams(even though the concentration in cerebrospinal fluid is only 5% of theserum level) (Gerber et al., Antimicrob Agents Chemother, 2001,45:2169-2172, incorporated by reference herein in its entirety; Cabelloset al., Antimicrob Agents Chemother, 2003, 47:1907-1911, incorporated byreference herein in its entirety); in models of central venouscatheter-associated infection by vancomycin-resistant Enterococcusfaecium (Rupp et al., J Antimicrob Chemother 2001, 47:705-707,incorporated by reference herein in its entirety); and in models ofendocarditis caused by vancomycin-susceptible or -resistant Enterococcusfaecalis (Lefort et al., Antimicrob Agents Chemother, 2000,44:3017-3021, incorporated by reference herein in its entirety).Pharmacodynamic studies in a neutropenic mouse thigh model of S. aureusinfection suggested that the parameter that best predicts oritavancinefficacy is the ratio between the free Cmax concentration and theminimal inhibitory concentration (MIC) of the offending organism (freeCmax/MIC) (Boylan, C. et al., Antimicrob Agents Chemother, 2003,47:1700-1706). Additional favorable pharmacodynamic characteristicsinclude prolonged post-antibiotic effects, and synergy with β-lactams oraminoglycosides (Lefort et al., Antimicrob Agents Chemother, 2000,44:3017-3021, incorporated by reference herein; Baltch et al.,Antimicrob Agents Chemother 1998, 42:2564-2568, incorporated byreference herein in its entirety).

Accordingly, oritavancin can be classified as a highlyconcentration-dependent bactericidal antibiotic with prolongedpersistent effects, in the same way as aminoglycosides and, to someextent, quinolones (Craig, Infect Dis Clin North Am, 2003, 17:479-501,incorporated by reference herein in its entirety). This pharmacodynamicprofile contrasts with that of conventional glycopeptides for whichefficacy relies mainly upon the area under the curve/MIC ratio, becausethey show time-dependent activity and persistent effects (Craig, InfectDis Clin North Am, 2003, 17:479-501, incorporated by reference herein inits entirety).

One pharmacokinetic property of oritavancin is its prolonged retentionin the organism, which destines it to a once-a-day scheme ofadministration. The exceptionally long terminal half-life suggests theexistence of storage sites within the organism. Studies on culturedmacrophages indicated that the drug accumulates slowly (by an endocyticprocess) but importantly in the lysosomes, from which its efflux isextremely slow. This explains why it is bactericidal againstintracellular forms of Staphylococcus or Enterococcus infections, butnot against cytosolic bacteria such as Listeria monocytogenes (Al Nawaset al., Infection 2000, 28:214-218, incorporated by reference herein inits entirety; Seral et al., Antimicrob Agents Chemother, 2003,47:2283-2292v). Corroborating these data, a recent study in volunteersdemonstrated that oritavancin reaches high concentrations not only inepithelial lining fluid but also in alveolar macrophages (Rodvold etal., Clin Microbiol Infect 2004, incorporated by reference herein in itsentirety).

According to one embodiment, the glycopeptide antibiotic of the presentinvention is telavancin (TD-6424). Telavancin is a semi-syntheticderivative of vancomycin, possessing a hydrophobic side chain on thevancosamine sugar (decylaminoethyl) and a (phosphonomethyl) aminomethylsubstituant on the cyclic peptidic core (FIG. 3; van Bambeke, F., Curr.Opin. Pharm., 4(5): 471-478; Judice, J. et al., Bioorg Med Chem Lett2003, 13: 4165-4168, incorporated by reference herein in its entirety).The length of the hydrophobic side chain was chosen to reach acompromise between optimized activity against MRSA (8-10 carbons) andVanA enterococci (12-16 carbons). Pharmacological studies suggest thatthe enhanced activity of telavancin on S. pneumoniae, S. aureus (to alesser extent), and staphylococci or enterococci harboring the vanA genecluster results from a complex mechanism of action which, on the basisof data obtained with close analogs, involves a perturbation of lipidsynthesis and possibly membrane disruption.

The polar substituent introduced on the resorcinol moiety improves thedistribution of the molecule in the body and counterbalances theprolonging effect of the lipophilic side chain on the half-life, whichis now approximately 7 h and still compatible with a once-dailyadministration. Pharmacodynamic properties include a prolongedpost-antibiotic effect and a concentration-dependent bactericidalactivity; therefore, one would propose to calculate the pharmacodynamicbreakpoint on the basis of the free C_(max)/MIC ratio, as done fororitavancin.

According to one embodiment, the glycopeptide antibiotic of the presentinvention is dalbavancin (BI 397). Dalbavancin is a semi-syntheticderivative of A40926, a glycopeptide with a structure related to that ofteicoplanin (FIG. 3; Malabarba et al., Curr Med Chem 2001, 8:1759-1773,incorporated by reference herein in its entirety; Malabarba et al., JAntibiot (Tokyo) 1994, 47:1493-1506, incorporated by reference herein inits entirety).

As with oritavancin and telavancin, dalbavancin is more active againstS. pneumoniae than are conventional glycopeptides, and its activityagainst S. aureus is also substantially improved, which was not observedwith the semi-synthetic derivatives of vancomycin. However, studies haveshown that it is not more active than teicoplanin against enterococciharboring the VanA phenotype of resistance to glycopeptides. Dalbavancinis also characterized by a marked bactericidal character and a synergismwith penicillin. The pharmacodynamic breakpoint calculated (as for theother bactericidal glycopeptides) on the basis of the free C_(max)/MICratio is of the same order of magnitude. Pharmacokinetic parameters andpharmacodynamic breakpoints for glycopeptides at doses pertinent totheir use in humans (or the foreseen doses for molecules in development)are shown in Table 3. Dalbavancin showed such a prolonged half-life thatits plasma concentration exceeds the minimal bactericidal concentrationof target organisms even at one week after administration of a single1000 mg dose; free levels, however, are close to the MICs at theseconditions (Steiert, M. et al., Curr Opin Investig Drugs 2002,3:229-233, incorporated by reference herein in its entirety). Theseresults indicate that a single dose of dalbavancin significantly reducesthe bacterial load in animal models of granuloma pouch infection by MRSA(Jabes et al., Antimicrob Agents Chemother 2004, 48:1118-1123,incorporated by reference herein in its entirety), endocarditis byvancomycin-susceptible or -intermediate staphylococci (Lefort et al.,Antimicrob Agents Chemother 2004, 48:1061-1064, incorporated byreference herein in its entirety), or pneumonia by penicillin-resistantpneumococci (Candiani et al., 41th Interscience Conference onAntimicrobial Agents and Chemotherapy 2001, Chicago, Ill. [Abstract989], incorporated by reference herein in its entirety).

TABLE 3 Pharmacokinetic parameters and pharmacodynamic breakpoints forglycopeptides (van Bambeke, Curr. Opin. Pharm., 2004, 4: 471-78,incorporated by reference herein) Glycopeptide and dosage VancomycinOritavancin Telavancin Teicoplanin Dalbavanc in Parameter (units) (15mg/kg) (3 mg/kg) (7.5 mg/kg) (6 mg/kg) (15 mg/kg) C_(max) (mg/L) 20-5031 89 43 312 V_(d) (L/kg) 0.3 0.1 0.9-1.6 0.11 Protein binding (%) 10-5590 90-93 90 98 Terminal half-life 4-8 360 7  83-168 149 (h) AUC (mg ·h/L) 260 152 600 550 27103 PD breakpoint based 2 (15 mg/kg 0.1 (3 mg/kg)0.5 0.4 (6 mg/kg), 4 on (free AUC)/MIC twice-daily) 0.3 (10 mg/kg) ratioPD breakpoint based 0.3 (3 mg/kg) 1 0.6 on (free C_(max)/MIC 1 (10mg/kg) ratio

According to one embodiment, the glycopeptide antibiotic if the presentinvention is conjugated to an amino acid or a derivative thereof. In asfurther embodiment, conjugation of the glycopeptides is to the Nterminus or C terminus of the amino acid or derivative thereof. Inanother embodiment, the glycopeptides antibiotic is conjugated to theamino acid side chain.

In one embodiment, the conjugation of the amino acid or derivativethereof to the glycopeptide antibiotic forms a stabilized glycopeptideantibiotic-amino acid complex. In one embodiment, the stabilizedglycopeptide antibiotic-amino acid complex is associated with a lipid.For example, in one embodiment, the lipid is conjugated (e.g., bound) tothe glycopeptide antibiotic-amino acid complex. In one embodiment, theglycopeptide antibiotic-amino acid complex is entrapped by the lipidcomponent, for example, where the lipid is in the form of a liposome orplurality of liposomes.

In one embodiment, the stabilized glycopeptide antibiotic composition ofthe described invention includes an amino acid or derivative thereof. Inone embodiment, the amino acid or derivative thereof is selected fromthe group consisting of alanine (ALA), D-alanine (D-ALA),alanine-alanine (ALA-ALA), beta-alanine (bALA), 3-aminobutanoic acid(3-ABA), gamma-aminobutyric acid (GABA), glutamic acid (GLU), D-glutamicacid (D-GLU), glycine (GLY), glycylglycine (GLY-GLY), aspartic acid(ASP), D-aspartic acid (D-ASP), bicine, tricine, sarcosine,iminodiacetic acid (IDAA), and combinations thereof. Structures ofexemplary amino acids or derivatives thereof useful in the invention areshown below in Table 4.

TABLE 4 Structure of exemplary amino acids and derivatives thereof Aminoacid/Amino Acid Derivative Structure D-ALA D-Alanine

ASP Aspartic acid

Bicine

D-GLU D-Glutamic acid

GLY-GLY Glycylglycine

IDAA Iminodiacetic acid

According to one embodiment, the stabilized lipid-based glycopeptideantibiotic composition maintains at least 98.0% of its originalbiological activity for at least 200 days at 4° C. According to anotherembodiment, the stabilized lipid-based glycopeptide antibioticcomposition maintains at least 98.5% of its original biological activityfor at least 150 days at 4° C. According to another embodiment, thestabilized lipid-based glycopeptide antibiotic composition maintains atleast 99.0% of its original biological activity for at least 100 days at4° C. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition maintains at least 99.5% of itsoriginal biological activity for at least 50 days at 4° C. According toanother embodiment, the stabilized lipid-based glycopeptide antibioticcomposition maintains at least 99.9% of its original biological activityfor at least two weeks at 4° C.

According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition maintains at least 80% of its original biologicalactivity for at least 200 days at room temperature (RT).

According to one embodiment, the stabilized lipid-based glycopeptideantibiotic composition of the present invention produces productdegradants crystalline at a rate less than 0.05% by weight per week at4° C. According to a further embodiment, the stabilized lipid-basedglycopeptide antibiotic composition produces product degradants at arate less than 0.04% by weight per week at 4° C. According to a furtherembodiment, the stabilized lipid-based glycopeptide antibioticcomposition produces product degradants at a rate less than 0.03% byweight per week at 4° C. According to a further embodiment, thestabilized lipid-based glycopeptide antibiotic composition producesproduct degradants at a rate less than 0.02% by weight per week at 4° C.According to a further embodiment, the stabilized lipid-basedglycopeptide antibiotic composition produces product degradants at arate less than 0.01% by weight per week at 4° C. In one embodiment, theproduct degradants produced at 4° C. are crystalline degradationproducts (i.e., CDP-I-m plus CDP-I-M)

According to one embodiment, the stabilized lipid-based glycopeptideantibiotic composition of the present invention produces productdegradants at a rate less than about 0.5% by weight per week at roomtemperature. According to a further embodiment, the stabilizedlipid-based glycopeptide antibiotic composition of the present inventionproduces product degradants at a rate less than about 0.4% by weight perweek at room temperature. According to a further embodiment, thestabilized lipid-based glycopeptide antibiotic composition of thepresent invention produces product degradants at a rate less than about0.3% by weight per week at room temperature. According to a yet furtherembodiment, the stabilized lipid-based glycopeptide antibioticcomposition of the present invention produces product degradants at arate less than about 0.2% by weight per week at room temperature. In oneembodiment, the product degradants produced at room temperature arecrystalline degradation products (i.e., CDP-I-m plus CDP-I-M)

In one embodiment, the stabilized lipid-based glycopeptide antibioticcomposition comprising a lipid component, a glycopeptide antibioticcomponent, and an amino acid or derivative thereof is at least 44% morestable than a lipid-based glycopeptide antibiotic that does not comprisean amino acid or derivative thereof. In a further embodiment, thestabilized lipid-based glycopeptide antibiotic composition comprising alipid component, a glycopeptide antibiotic component, and an amino acidor derivative thereof is at least 55% more stable than a lipid-basedglycopeptide antibiotic that does not comprise an amino acid orderivative thereof. In a further embodiment, the stabilized lipid-basedglycopeptide antibiotic composition comprising a lipid component, aglycopeptide antibiotic component, and an amino acid or derivativethereof is at least 66% more stable than a lipid-based glycopeptideantibiotic that does not comprise an amino acid or derivative thereof.In a further embodiment, the stabilized lipid-based glycopeptideantibiotic composition comprising a lipid component, a glycopeptideantibiotic component, and an amino acid or derivative thereof is atleast 77% more stable than a lipid-based glycopeptide antibiotic thatdoes not comprise an amino acid or derivative thereof. In a yet furtherembodiment, the stabilized lipid-based glycopeptide antibioticcomposition comprising a lipid component, a glycopeptide antibioticcomponent, and an amino acid or derivative thereof is at least 88% morestable than a lipid-based glycopeptide antibiotic that does not comprisean amino acid or derivative thereof.

According to another embodiment, the molar ratio of the glycopeptideantibiotic to the amino acid or amino acid derivative ranges from about1:1 to about 1:4. According to another embodiment, the molar ratio ofthe glycopeptide antibiotic to the amino acid or amino acid derivativeranges from about 1:1 to about 1:3. According to another embodiment, themolar ratio of the glycopeptide antibiotic to the amino acid or aminoacid derivative ranges from about 1:1 to about 1:2. According to anotherembodiment, the molar ratio of the glycopeptide antibiotic to the aminoacid or amino acid derivative is about 1:4. According to anotherembodiment, the molar ratio of the glycopeptide antibiotic to the aminoacid or amino acid derivative is about 1:3. According to anotherembodiment, the molar ratio of the glycopeptide antibiotic to the aminoacid or amino acid derivative is about 1:2. According to anotherembodiment, the molar ratio of the glycopeptide antibiotic to the aminoacid or amino acid derivative is about 1:1.

According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.0 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.1 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.2 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.3 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.4 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.5 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.6 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.7 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.8 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 5.9 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 6.0 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 6.1 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 6.2 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 6.3 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH ranging from about 6.4 to about 6.5.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition has a pH of 5.0. According to another embodiment,the stabilized lipid-based glycopeptide antibiotic composition has a pHof 5.5. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition has a pH of 6.0. According toanother embodiment, the stabilized lipid-based glycopeptide antibioticcomposition has a pH of 6.5.

According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 20 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 30 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 40 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 50 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 60 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 70 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 80 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 90 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 100 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 110 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 120 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 130 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 140 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 150 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 160 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 170 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 180 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition ranges from 190 mg/mL to 200 mg/mL.According to another embodiment, the concentration of the glycopeptideantibiotic in the composition is 200 mg/mL. According to anotherembodiment, the concentration of the glycopeptide antibiotic in thecomposition is 100 mg/mL.

According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition of the present invention comprises an aqueousdispersion of liposomes. The formulation can contain lipid excipients toform the liposomes, and salts/buffers to provide the appropriateosmolarity and pH. The formulation can comprise a pharmaceuticalexcipient. The pharmaceutical excipient can be a liquid, diluent,solvent or encapsulating material, involved in carrying or transportingany subject composition or component thereof from one organ, or portionof the body, to another organ, or portion of the body. Each excipientmust be “acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient.Suitable excipients include trehalose, raffinose, mannitol, sucrose,leucine, trileucine, and calcium chloride. Examples of other suitableexcipients include (1) sugars, such as lactose, and glucose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, and polyethyleneglycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar;(14) buffering agents, such as magnesium hydroxide and aluminumhydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonicsaline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphatebuffer solutions; and (21) other non-toxic compatible substancesemployed in pharmaceutical formulations.

Representative lipid-based glycopeptide antibiotic formulations areshown in Table 5.

TABLE 5 Representative formulation components Glycopeptide LipidComponent Amino Acid Vancomycin DPPC Bicine Vancomycin DPPC GLUVancomycin DPPC GLY-GLY Vancomycin DPPC IDAA Vancomycin DPPC ASPVancomycin DPPC D-ALA Vancomycin DPPC + Cholesterol Bicine VancomycinDPPC + Cholesterol GLU Vancomycin DPPC + Cholesterol GLY-GLY VancomycinDPPC + Cholesterol IDAA Vancomycin DPPC + Cholesterol ASP VancomycinDPPC + Cholesterol D-ALA Vancomycin DPPG Bicine Vancomycin DPPG GLUVancomycin DPPG GLY-GLY Vancomycin DPPG IDAA Vancomycin DPPG ASPVancomycin DPPG D-ALA Vancomycin DPPG + Cholesterol Bicine VancomycinDPPG + Cholesterol GLU Vancomycin DPPG + Cholesterol GLY-GLY VancomycinDPPG + Cholesterol IDAA Vancomycin DPPG + Cholesterol ASP VancomycinDPPG + Cholesterol D-ALA Vancomycin DPPC + DPPG + Cholesterol BicineVancomycin DPPC + DPPG + Cholesterol GLU Vancomycin DPPC + DPPG +Cholesterol GLY-GLY Vancomycin DPPC + DPPG + Cholesterol IDAA VancomycinDPPC + DPPG + Cholesterol ASP Vancomycin DPPC + DPPG + Cholesterol D-ALAVancomycin POPC Bicine Vancomycin POPC GLU Vancomycin POPC GLY-GLYVancomycin POPC IDAA Vancomycin POPC ASP Vancomycin POPC D-ALA

In one embodiment, the present invention provides a stabilizedglycopeptides composition comprising a lipid component, a glycopeptidesantibiotic and an amino acid or derivative thereof. In one embodiment,the lipid to glycopeptide molar ratio is 10:1 or less, 9:1 or less, 8:1or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less, 3:1 orless, 2:1 or less, 1:1 or less, 0.75:1 or less or 0.5:1 or less. Inanother embodiment, the lipid to glycopeptide molar ratio is about 1:1,about 2:1, about 3:1, about 4:1 or about 5:1.

In one embodiment, the present invention relates to a stabilizedlipid-glycopeptide antibiotic composition comprising a lipid component,a glycopeptide antibiotic, and an amino acid or derivative thereof,wherein the weight ratio of the total lipid component to theglycopeptide antibiotic is from about 0.1:1 to about 5:1. In a furtherembodiment, the weight ratio of the lipid component to the glycopeptideantibiotic is about 3:1 or less. In a further embodiment, the weightratio of the lipid component to the glycopeptide antibiotic is about 1:1or less. In another embodiment, the weight ratio of the lipid componentto the glycopeptide antibiotic is less than 1:1.

It is understood that for the ranges of values provided above, eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Representative lipid-based glycopeptide antibiotic formulations withexemplary lipid to glycopeptide ratios are shown in Table 6.

TABLE 6 Representative formulations with lipid to drug ratiosGlycopeptide Antibiotic Lipid Component Amino Acid Lipid:glycopeptideVancomycin DPPC Bicine 5:1 Vancomycin DPPC GLU 5:1 Vancomycin DPPCGLY-GLY 5:1 Vancomycin DPPC IDAA 5:1 Vancomycin DPPC ASP 5:1 VancomycinDPPC D-ALA 5:1 Vancomycin DPPC + Cholesterol Bicine 5:1 VancomycinDPPC + Cholesterol GLU 5:1 Vancomycin DPPC + Cholesterol GLY-GLY 5:1Vancomycin DPPC + Cholesterol IDAA 5:1 Vancomycin DPPC + Cholesterol ASP5:1 Vancomycin DPPC + Cholesterol D-ALA 5:1 Vancomycin DPPG Bicine 5:1Vancomycin DPPG GLU 5:1 Vancomycin DPPG GLY-GLY 5:1 Vancomycin DPPG IDAA5:1 Vancomycin DPPG ASP 5:1 Vancomycin DPPG D-ALA 5:1 Vancomycin DPPG +Cholesterol Bicine 5:1 Vancomycin DPPG + Cholesterol GLU 5:1 VancomycinDPPG + Cholesterol GLY-GLY 5:1 Vancomycin DPPG + Cholesterol IDAA 5:1Vancomycin DPPG + Cholesterol ASP 5:1 Vancomycin DPPG + CholesterolD-ALA 5:1 Vancomycin DPPC + DPPG + Bicine 5:1 Cholesterol VancomycinDPPC + DPPG + GLU 5:1 Cholesterol Vancomycin DPPC + DPPG + GLY-GLY 5:1Cholesterol Vancomycin DPPC + DPPG + IDAA 5:1 Cholesterol VancomycinDPPC + DPPG + ASP 5:1 Cholesterol Vancomycin DPPC + DPPG + D-ALA 5:1Cholesterol Vancomycin POPC Bicine 5:1 Vancomycin POPC GLU 5:1Vancomycin POPC GLY-GLY 5:1 Vancomycin POPC IDAA 5:1 Vancomycin POPC ASP5:1 Vancomycin POPC D-ALA 5:1 Vancomycin DPPC Bicine 3:1 Vancomycin DPPCGLU 3:1 Vancomycin DPPC GLY-GLY 3:1 Vancomycin DPPC IDAA 3:1 VancomycinDPPC ASP 3:1 Vancomycin DPPC D-ALA 3:1 Vancomycin DPPC + CholesterolBicine 3:1 Vancomycin DPPC + Cholesterol GLU 3:1 Vancomycin DPPC +Cholesterol GLY-GLY 3:1 Vancomycin DPPC + Cholesterol IDAA 3:1Vancomycin DPPC + Cholesterol ASP 3:1 Vancomycin DPPC + CholesterolD-ALA 3:1 Vancomycin DPPG Bicine 3:1 Vancomycin DPPG GLU 3:1 VancomycinDPPG GLY-GLY 3:1 Vancomycin DPPG IDAA 3:1 Vancomycin DPPG ASP 3:1Vancomycin DPPG D-ALA 3:1 Vancomycin DPPG + Cholesterol Bicine 3:1Vancomycin DPPG + Cholesterol GLU 3:1 Vancomycin DPPG + CholesterolGLY-GLY 3:1 Vancomycin DPPG + Cholesterol IDAA 3:1 Vancomycin DPPG +Cholesterol ASP 3:1 Vancomycin DPPG + Cholesterol D-ALA 3:1 VancomycinDPPC + DPPG + Bicine 3:1 Cholesterol Vancomycin DPPC + DPPG + GLU 3:1Cholesterol Vancomycin DPPC + DPPG + GLY-GLY 3:1 Cholesterol VancomycinDPPC + DPPG + IDAA 3:1 Cholesterol Vancomycin DPPC + DPPG + ASP 3:1Cholesterol Vancomycin DPPC + DPPG + D-ALA 3:1 Cholesterol VancomycinPOPC Bicine 3:1 Vancomycin POPC GLU 3:1 Vancomycin POPC GLY-GLY 3:1Vancomycin POPC IDAA 3:1 Vancomycin POPC ASP 3:1 Vancomycin POPC D-ALA3:1 Vancomycin DPPC Bicine 2:1 Vancomycin DPPC GLU 2:1 Vancomycin DPPCGLY-GLY 2:1 Vancomycin DPPC IDAA 2:1 Vancomycin DPPC ASP 2:1 VancomycinDPPC D-ALA 2:1 Vancomycin DPPC + Cholesterol Bicine 2:1 VancomycinDPPC + Cholesterol GLU 2:1 Vancomycin DPPC + Cholesterol GLY-GLY 2:1Vancomycin DPPC + Cholesterol IDAA 2:1 Vancomycin DPPC + Cholesterol ASP2:1 Vancomycin DPPC + Cholesterol D-ALA 2:1 Vancomycin DPPG Bicine 2:1Vancomycin DPPG GLU 2:1 Vancomycin DPPG GLY-GLY 2:1 Vancomycin DPPG IDAA2:1 Vancomycin DPPG ASP 2:1 Vancomycin DPPG D-ALA 2:1 Vancomycin DPPG +Cholesterol Bicine 2:1 Vancomycin DPPG + Cholesterol GLU 2:1 VancomycinDPPG + Cholesterol GLY-GLY 2:1 Vancomycin DPPG + Cholesterol IDAA 2:1Vancomycin DPPG + Cholesterol ASP 2:1 Vancomycin DPPG + CholesterolD-ALA 2:1 Vancomycin DPPC + DPPG + Bicine 2:1 Cholesterol VancomycinDPPC + DPPG + GLU 2:1 Cholesterol Vancomycin DPPC + DPPG + GLY-GLY 2:1Cholesterol Vancomycin DPPC + DPPG + IDAA 2:1 Cholesterol VancomycinDPPC + DPPG + ASP 2:1 Cholesterol Vancomycin DPPC + DPPG + D-ALA 2:1Cholesterol Vancomycin POPC Bicine 2:1 Vancomycin POPC GLU 2:1Vancomycin POPC GLY-GLY 2:1 Vancomycin POPC IDAA 2:1 Vancomycin POPC ASP2:1 Vancomycin POPC D-ALA 2:1 Vancomycin DPPC Bicine 1:1 Vancomycin DPPCGLU 1:1 Vancomycin DPPC GLY-GLY 1:1 Vancomycin DPPC IDAA 1:1 VancomycinDPPC ASP 1:1 Vancomycin DPPC D-ALA 1:1 Vancomycin DPPC + CholesterolBicine 1:1 Vancomycin DPPC + Cholesterol GLU 1:1 Vancomycin DPPC +Cholesterol GLY-GLY 1:1 Vancomycin DPPC + Cholesterol IDAA 1:1Vancomycin DPPC + Cholesterol ASP 1:1 Vancomycin DPPC + CholesterolD-ALA 1:1 Vancomycin DPPG Bicine 1:1 Vancomycin DPPG GLU 1:1 VancomycinDPPG GLY-GLY 1:1 Vancomycin DPPG IDAA 1:1 Vancomycin DPPG ASP 1:1Vancomycin DPPG D-ALA 1:1 Vancomycin DPPG + Cholesterol Bicine 1:1Vancomycin DPPG + Cholesterol GLU 1:1 Vancomycin DPPG + CholesterolGLY-GLY 1:1 Vancomycin DPPG + Cholesterol IDAA 1:1 Vancomycin DPPG +Cholesterol ASP 1:1 Vancomycin DPPG + Cholesterol D-ALA 1:1 VancomycinDPPC + DPPG + Bicine 1:1 Cholesterol Vancomycin DPPC + DPPG + GLU 1:1Cholesterol Vancomycin DPPC + DPPG + GLY-GLY 1:1 Cholesterol VancomycinDPPC + DPPG + IDAA 1:1 Cholesterol Vancomycin DPPC + DPPG + ASP 1:1Cholesterol Vancomycin DPPC + DPPG + D-ALA 1:1 Cholesterol VancomycinPOPC Bicine 1:1 Vancomycin POPC GLU 1:1 Vancomycin POPC GLY-GLY 1:1Vancomycin POPC IDAA 1:1 Vancomycin POPC ASP 1:1 Vancomycin POPC D-ALA1:1

According to another aspect, the described invention provides a methodfor preparing a stabilized lipid-based glycopeptide antibioticcomposition, wherein the method comprises:

infusing, in an in-line fashion, a first stream of a lipid solutioncontaining a lipid component in a solvent with a second stream of anaqueous solution comprising a glycopeptide antibiotic and an amino acidor a derivative thereof, wherein the amino acid or the derivativethereof binds to the glycopeptide antibiotic and forms a stabilizedglycopeptide antibiotic-amino acid complex, and the stabilizedglycopeptide antibiotic-amino acid complex is entrapped by or complexedwith the a lipid component (i.e., one lipid or a mixture of multiplelipids).

According to some embodiments, the first stream in step (a) contains alipid dissolved in a solvent. According to some such embodiments, thesolvent is ethanol.

According to another embodiment, the lipid solution comprises from about2 mg/mL to 200 mg/mL of lipids. According to another embodiment, thelipid solution comprises about 20 mg/mL of lipids.

Examples of the lipid component that can be used in preparing thestabilized lipid-based glycopeptide antibiotic composition of thepresent invention includes, but is limited to, phosphatidylcholine (PC),phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine(PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), eggphosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), eggphosphatidylinositol (EPI), egg phosphatidylserine (EPS),phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soyphosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soyphosphatidylserine (SPS), soy phosphatidylinositol (SPI), soyphosphatidylethanolamine (SPE), soy phosphatidic acid (SPA),hydrogenated egg phosphatidylcholine (HEPC), hydrogenated eggphosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol(HEPI), hydrogenated egg phosphatidylserine (HEPS), hydrogenatedphosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA),hydrogenated soy phosphatidylcholine (HSPC), hydrogenated soyphosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS),hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soyphosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid(HSPA), dipalmitoylphosphatidylcholine (DPPC),dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol(DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), di stearoylphosphatidylglycerol(DSPG), dioleoylphosphatidylcholine (DOPC),dioleylphosphatidylethanolamine (DOPE),palmitoylstearoylphosphatidyl-choline (PSPC),palmitoylstearolphosphatidylglycerol (PSPG),mono-oleoyl-phosphatidylethanolamine (MOPE), tocopherol, tocopherol hemisuccinate, cholesterol sulfate, cholesteryl hemisuccinate, cholesterolderivatives, ammonium salts of fatty acids, ammonium salts ofphospholipids, ammonium salts of glycerides, myristylamine,palmitylamine, laurylamine, stearylamine, dilauroyl ethylphosphocholine(DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoylethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP),N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP), di stearoylphosphatidylglycerol (DSPG),dimyristoylphosphatidylacid (DMPA), dipalmitoylphosphatidylacid (DPPA),di stearoylphosphatidylacid (DSPA), dimyristoylphosphatidylinositol(DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol (DSPI), dimyristoylphosphatidylserine(DMPS), dipalmitoylphosphatidylserine (DPPS),distearoylphosphatidylserine (DSPS), or a mixture thereof.

According to another embodiment, the lipid component that can be used inpreparing a stabilized lipid-based glycopeptide antibiotic compositionof the present invention includes mixed phospholipids, e.g.,palmitoylstearoylphosphatidylcholine (PSPC) andpalmitoylstearoylphosphatidylglycerol (PSPG)), triacylglycerol,diacylglycerol, seranide, sphingosine, sphingomyelin, and singleacylated phospholipids, such as mono-oleoyl-phosphatidylethanol amine(MOPE).

According to another embodiment, the lipid component that can be used inpreparing the stabilized lipid-based glycopeptide antibiotic compositionof the present invention includes ammonium salts of fatty acids,phospholipids and glycerides, sterols, phosphatidylglycerols (PGs),phosphatidic acids (PAs), phosphotidylcholines (PCs),phosphatidylinositols (PIs) and the phosphatidylserines (PSs). The fattyacids include fatty acids of carbon chain lengths of 12 to 26 carbonatoms that are either saturated or unsaturated. Some specific examplesinclude, but are not limited to, myristylamine, palmitylamine,laurylamine and stearylamine, dilauroyl ethylphosphocholine (DLEP),dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine(DPEP) and distearoyl ethylphosphocholine (DSEP),N-(2,3-di-(9(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA) and 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP).

According to another embodiment, the lipid component consistsessentially of a phosphatidylcholine. According to another embodiment,the lipid component consists essentially ofdipalmitoylphosphatidylcholine (DPPC). According to another embodiment,the lipid component consists essentially ofpalmitoyloleoylphosphatidylcholine (POPC).

According to another embodiment, the lipid component consistsessentially of phosphatidylglycerol. According to another embodiment,the lipid component consists essentially of1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG).

According to another embodiment, the lipid component includes a sterol,including, but not limited to, cholesterol and ergosterol. In oneembodiment, the lipid component consists of a sterol and one additionallipid. In a further embodiment, the sterol is cholesterol.

According to another embodiment, the first flow rate is between 0.1L/min and 100 L/min. According to another embodiment, the first flowrate is between 0.5 L/min and 10 L/min. According to another embodiment,the first flow rate is between 0.5 L/min and 1.5 L/min. According toanother embodiment, the first flow rate is about 10 L/min. According toanother embodiment, the first flow rate is about 1 L/min.

According to another embodiment, the molar ratio of the glycopeptideantibiotic to the amino acid or amino acid derivative ranges from about1:1 to about 1:4. According to another embodiment, the molar ratio ofthe glycopeptide antibiotic to the amino acid or amino acid derivativeranges from about 1:1 to about 1:3. According to another embodiment, themolar ratio of the glycopeptide antibiotic to the amino acid or aminoacid derivative ranges from about 1:1 to about 1:2. According to anotherembodiment, the molar ratio of the glycopeptide antibiotic to the aminoacid or amino acid derivative is about 1:4. According to anotherembodiment, the molar ratio of the glycopeptide antibiotic to the aminoacid or amino acid derivative is about 1:3. According to anotherembodiment, the molar ratio of the glycopeptide antibiotic to the aminoacid or amino acid derivative is about 1:2. According to anotherembodiment, the molar ratio of the glycopeptide antibiotic to the aminoacid or amino acid derivative is about 1:1.

According to another embodiment, the aqueous solution comprises betweenabout 20 mg/mL and about 500 mg/mL of the glycopeptide antibiotic.According to another embodiment, the first stream comprises betweenabout 50 mg/mL and about 250 mg/mL of the glycopeptide antibiotic.According to another embodiment, the first stream comprises betweenabout 100 mg/mL and about 200 mg/mL of the glycopeptide antibiotic.According to another embodiment, the first stream comprises about 100mg/mL of the glycopeptide antibiotic. According to another embodiment,the first stream comprises about 200 mg/mL of the glycopeptideantibiotic.

According to another embodiment, the aqueous solution has pH of from 5.0to 6.5. According to another embodiment, the aqueous solution has pH offrom 5.1 to 6.5. According to another embodiment, the aqueous solutionhas pH of from 5.2 to 6.5. According to another embodiment, the aqueoussolution has pH of from 5.3 to 6.5. According to another embodiment, theaqueous solution has pH of from 5.4 to 6.5. According to anotherembodiment, the aqueous solution has a pH ranging from 5.5 to 6.5.According to another embodiment, the aqueous solution has pH of from 5.6to 6.5. According to another embodiment, the aqueous solution has pH offrom 5.7 to 6.5. According to another embodiment, the aqueous solutionhas pH of from 5.8 to 6.5. According to another embodiment, the aqueoussolution has pH of from 5.9 to 6.5. According to another embodiment, theaqueous solution has pH of from 6.0 to 6.5. According to anotherembodiment, the aqueous solution has pH of from 6.1 to 6.5. According toanother embodiment, the aqueous solution has pH of from 6.2 to 6.5.According to another embodiment, the aqueous solution has pH of from 6.3to 6.5. According to another embodiment, the aqueous solution has pH offrom 6.4 to 6.5. According to another embodiment, the aqueous solutionhas a pH of 5.0. According to another embodiment, the aqueous solutionhas a pH of 5.5. According to another embodiment, the aqueous solutionhas a pH of 6.0. According to another embodiment, the aqueous solutionhas a pH of 6.5.

According to one embodiment, the second flow rate is between 0.1 L/minand 100 L/min. According to another embodiment, the second flow rate isbetween 0.5 L/min and 10 L/min. According to another embodiment, thesecond flow rate is between 0.5 L/min and 2 L/min. According to anotherembodiment, the second flow rate is between 1 L/min and 2 L/min.According to another embodiment, the second flow rate is about 1.5L/min. According to another embodiment, the second flow rate is about 10L/min.

According to one embodiment, the ratio of the second flow rate to thefirst flow rate is from about 0.1:1.0 to about 1:0.1.0. According toanother embodiment, the ratio of the second flow rate to the first flowrate is from about 0.5:1.0 to about 2.0:1.0. According to anotherembodiment, the ratio of the second flow rate to the first flow rate isfrom about 1.0:1.0 to about 2.0:1.0. According to one embodiment, theratio of the second flow rate to the first flow rate is about 1.5:1.0.

According to another embodiment, the saline solution comprises fromabout 0.9% wt/wt to about 1.5% wt/wt of sodium chloride (NaCl).According to another embodiment, the saline solution comprises less than0.9% wt/wt of sodium chloride (NaCl). According to another embodiment,the saline solution comprises about 0.9% wt/wt of sodium chloride(NaCl). According to another embodiment, the saline solution comprisesfrom about 1.0% wt/wt to about 1.5% wt/wt of sodium chloride (NaCl).According to another embodiment, the saline solution comprises from 1.1%wt/wt to 1.5% wt/wt of sodium chloride (NaCl). According to anotherembodiment, the saline solution comprises from about 1.2% wt/wt to about1.5% wt/wt of sodium chloride (NaCl). According to another embodiment,the saline solution comprises from about 1.3% wt/wt to about 1.5% wt/wtof sodium chloride (NaCl). According to another embodiment, the salinesolution comprises from 1.4% wt/wt to about 1.5% wt/wt of sodiumchloride (NaCl). According to another embodiment, the saline solutioncomprises about 1.5% wt/wt of sodium chloride (NaCl). According toanother embodiment, the saline solution comprises at least 1.5% wt/wt ofsodium chloride (NaCl).

According to one embodiment, the saline solution flow rate is between0.1 L/min and 10 L/min. According to another embodiment, the salinesolution flow rate is between 0.5 L/min and 2 L/min. According toanother embodiment, the saline solution flow rate is about 1.5 L/min.According to another embodiment, the saline solution flow rate is about10 L/min.

According to another embodiment, the method produces an infused mixture,wherein the infused mixture comprises a first population of glycopeptideantibiotics entrapped with the lipid and a second population ofglycopeptide antibiotics unentrapped with the lipid.

According to another embodiment, the method of preparation furthercomprises washing the infused mixture comprising lipid-associated orlipid-unassociated glycopeptide antibiotics by infusing a washing feedat a fourth flow rate, wherein the washing feed comprises a salinesolution.

According to another embodiment, the washing step is carried outfollowing the formation of infused mixture, wherein the infused mixturecomprises a first population of glycopeptide antibiotics entrapped withthe lipid and a second population of glycopeptide antibioticsunentrapped with the lipid.

According to another embodiment, the saline solution comprises fromabout 0.9% wt/wt to about 1.5% wt/wt of sodium chloride (NaCl).According to another embodiment, the saline solution comprises 0.9%wt/wt of sodium chloride (NaCl). According to another embodiment, thesaline solution comprises from about 1.0% wt/wt to about 1.5% wt/wt ofsodium chloride (NaCl). According to another embodiment, the salinesolution comprises from 1.1% wt/wt to 1.5% wt/wt of sodium chloride(NaCl). According to another embodiment, the saline solution comprisesfrom about 1.2% wt/wt to about 1.5% wt/wt of sodium chloride (NaCl).According to another embodiment, the saline solution comprises fromabout 1.3% wt/wt to about 1.5% wt/wt of sodium chloride (NaCl).According to another embodiment, the saline solution comprises from 1.4%wt/wt to about 1.5% wt/wt of sodium chloride (NaCl). According toanother embodiment, the saline solution comprises 1.5% wt/wt of sodiumchloride (NaCl).

According to another embodiment, the method further comprises a step forconcentrating lipid-associated glycopeptide antibiotics in theformulation.

According to another aspect, the described invention provides a methodfor treating a bacterial pulmonary infection, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a stabilized lipid-based glycopeptide antibiotic composition,wherein the composition comprises:

(a) a lipid component, (b) a glycopeptide antibiotic component, and (c)an amino acid or a derivative thereof, wherein the amino acid orderivative thereof binds to the glycopeptide antibiotic and forms astabilized glycopeptide antibiotic-amino acid complex, and wherein thestabilized glycopeptide antibiotic-amino acid complex is entrapped bythe lipid.

According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition can treat infections caused by Gram-positivebacteria, including, but not limited to, genera Staphylococcus,Streptococcus, Enterococcus, Bacillus, Corynebacterium, Nocardia,Clostridium, and Listeria.

Staphylococci are Gram-positive spherical bacteria that occur inmicroscopic clusters resembling grapes. While there are 20 species ofStaphylococcus, only Staphylococcus aureus and Staphylococcus epidermisare known to be significant in their interactions with humans. S. aureuscolonizes mainly the nasal passages, but it may be found regularly inmost anatomical locales, including skin oral cavity, andgastrointestinal tract. S. epidermis is an inhabitant of the skin.Examples of Staphylcocci treatable with the lipid-based glycopeptideantibiotic composition of the present invention, include, but are notlimited to, S. aureus, S. auricularis, S. carnosus, S. epidermidis, S.haemolyticus, S. hyicus, S. intermedius, S. lugdunensis, S.saprophyticus, S. sciuri, S. simulans, and S. warneri.

Streptococci are Gram-positive, non-motile cocci that divide in oneplane, producing chains of cells. The Streptococci are a veryheterogeneous group of bacteria. The primary pathogens include S.pyrogenes and S. pneumoniae but other species can be opportunistic. S.pyrogenes is the leading cause of bacterial pharyngitis and tonsillitis.It can also produce sinusitis, otitis, arthritis, and bone infections.Some strains prefer skin, producing either superficial (impetigo) ordeep (cellulitis) infections. S. pneumoniae is the major cause ofbacterial pneumonia in adults. Its virulence is dictated by its capsule.Toxins produced by streptococci include: streptolysins (S & O), NADase,hyaluronidase, streptokinase, DNAses, erythrogenic toxin (which causesscarlet fever rash by producing damage to blood vessels; requires thatbacterial cells are lysogenized by phage that encodes toxin). Examplesof Streptococci treatable with the lipid-based glycopeptide antibioticcomposition of the present invention include, but are not limited to, S.agalactiae, S. anginosus, S. bovis, S. canis, S. constellatus, S.dysgalactiae, S. equi, S. equinus, S. iniae, S. intermedius, S. mitis,S. mutans, S. oralis, S. parasanguinis, S. peroris, S. pneumoniae, S.pyogenes, S. ratti, S. sahvarius, S. sahvarius ssp. thermophilus, S.sanguinis, S. sobrinus, S. suis, S. uberis, S. vestibularis, S.viridans, and S. zooepidemicus

The genus Enterococci consists of Gram-positive, facultatively anaerobicorganisms that are ovoid in shape and appear on smear in short chains,in pairs, or as single cells. Enterococci are important human pathogensthat are increasingly resistant to antimicrobial agents. These organismswere considered previously part of the genus Streptococcus but haverecently been reclassified into their own genus, called Enterococcus. Todate, 12 species pathogenic for humans have been described, includingthe most common human isolates, Enterococcus faecalis and Enterococcusfaecium. Enterococci cause between 5 and 15% of cases of endocarditis,which is treated best by the combination of a cell wall-active agent(such as penicillin or vancomycin) and an aminoglycoside to which theorganism is not highly resistant; this characteristically results in asynergistic bactericidal effect. Examples of Enterococci treatable withthe lipid-based glycopeptide antibiotic composition of the presentinvention include, but are not limited to, E. avium, E. durans, E.faecalis, E. faecium, E. gallinarum, and E. solitarius.

Bacteria of the genus Bacillus are aerobic, endospore-forming,gram-positive rods. The genus is one of the most diverse andcommercially useful groups of microorganisms. Representatives of thisgenus are distributed widely in soil, air, and water where they areinvolved in a range of chemical transformations. Some Bacillus speciesare known to cause health problems in humans. For example, Anthrax iscaused by Bacillus anthracis. Humans acquire the disease directly fromcontact with infected herbivores or indirectly via their products. Theclinical forms include (1) cutaneous anthrax, from handling infectedmaterial; (2) intestinal anthrax, from eating infected meat; and (3)pulmonary anthrax from inhaling spore-laden dust. Several other Bacillusspp., in particular B. cereus and to a lesser extent B. subtilis and B.licheniformis, are associated periodically with bacteremia/septicemia,endocarditis, meningitis, and infections of wounds, the ears, eyes,respiratory tract, urinary tract, and gastrointestinal tract. Bacilluscereus causes two distinct food poisoning syndromes: a rapid-onsetemetic syndrome characterized by nausea and vomiting, and a slower-onsetdiarrheal syndrome. Examples of pathogenic Bacillus species whoseinfection is treatable with the lipid-based glycopeptide antibioticcomposition of the present invention, include, but are not limited to,B. anthracis, B. cereus, and B. coagulans.

Corynebacteria are small, generally non-motile, Gram-positive, nonsporulating, pleomorphic bacilli. They are chemoorganotrophic, aerobic,or facultatively anaerobic, and they exhibit a fermentative metabolismunder certain conditions. Corybacterium diphtheriae is the etiologicalagent of diphtheria, an upper respiratory disease mainly affectingchildren. The virulence factors (i.e., diphtheria toxin) have beenstudied extensively. Examples of Corynebacterial species treatable withthe lipid-based glycopeptide antibiotic composition of the presentinvention include, for example, but are not limited to, Corynebacteriumdiphtheria, Corynebacterium pseudotuberculosis, Corynebacterium tenuis,Corynebacterium striatum, and Corynebacterium minutissimum.

The bacteria of the genus Nocardia are Gram-positive, partiallyacid-fast rods, which grow slowly in branching chains resembling fungalhyphae. Three species cause nearly all human infections: N. asteroides,N. brasiliensis, and N. caviae. Infection is by inhalation of airbornebacilli from an environmental source (soil or organic material); thedisease is not contagious. Skin lesions caused by N. brasiliensis oftenresult from direct inoculation. Nocardia subverts antimicrobialmechanisms of phagocytes, causing abscess or rarely granuloma formationwith hematogenous or lymphatic dissemination to the skin or centralnervous system. Examples of Nocardial species treatable with thelipid-based glycopeptide antibiotic composition of the presentinvention, include, for example, but are not limited to, N.aerocolonigenes, N. africana, N. argentinensis, N. asteroides, N. blackwellii, N. brasiliensis, N. brevicatena, N. carnea, N. caviae, N.cerradoensis, N. corallina, N. cyriacigeorgica, N. dassonvillei, N.elegans, N. farcinica, N. nigiitansis, N. nova, N. opaca, N.otitidis-cavarium, N. paucivorans, N. pseudobrasiliensis, N. rubra, N.transvelencesis, N. uniformis, N. vaccinii, and N. veterana.

Clostridia are spore-forming, Gram-positive, anaerobes. Clostridia arerod-shaped, but when producing spores they appear more like drumstickswith a bulge at one end. There are three species of clostridia thatcause widely recognized and often-deadly diseases. C. tetani is theetiological agent of tetanus, C. botulinum is the etiological agent ofbotulism, and C. perfringens is one of the etiological agents of gasgangrene. Tetanus is contracted through contact between spores of C.tetani and an open wound, such as stepping on a rusty nail. If ananaerobic environment is present, the spores will germinate. Tetanus isa neurological disease in which C. tetani releases an exotoxin calledtetanus toxin, which blocks the release of neurotransmitters from thepresynaptic membrane of inhibitory interneurons of spinal cord andbrainstem of mammals that regulate muscle contraction. Examples ofClostridium species treatable with the lipid-based glycopeptideantibiotic composition of the present invention, include, for example,but are not limited to, C. botulinum, C. tetani, C.difficile, C.perfringens, and C. sordellii.

Listeria are non spore-forming, nonbranching Gram-positive rods thatoccur individually or form short chains. Listeria are intracellularpathogens that use host-produced actin filaments for motility within thehost cell. L. monocytogenes is the causative agent of listeriosis. L.monocytogenes is a food-borne pathogen, which can survive normalrefrigeration processes. It can cause severe disease inimmunocompromised individuals, and pregnant women. Examples of Listeriaspecies treatable with the lipid-based glycopeptide antibioticcomposition of the present invention, include, for example, but are notlimited to, L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L.seeligeri, L. murrayi, and L. welshimeri.

Other Examples of Gram-positive bacterial species, whose infection istreatable with the lipid-based glycopeptide antibiotic composition ofthe present invention, include, but are not limited to,Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli,Klebsiella, Enterobacter, Serratia, Haemophilus, Yersinia pesos,Burkholderia pseudomallei, Burkholderia cepacia, Burkholderia gladioli,Burkholderia multivorans, Burkholderia vietnamiensis, Mycobacteriumtuberculosis, Mycobacterium avium complex (MAC) (Mycobacterium avium andMycobacterium intracellulare), Mycobacterium kansasii, Mycobacteriumxenopi, Mycobacterium marinum, Mycobacterium mucogenicum, Mycobacgeriumgordonae, Mycobacterium ulcerans, and Mycobacterium fortuitum complex(including, but not limited to, Mycrobacterium fortuitum, Mycrobacteriumperegrinum, Mycrobacterium chelonae, Mycrobacterium abscessus, andMycrobacterium mucogenicum.

Methicillin-resistant Staphyloccus aureus (MRSA) is a type ofstaphylococcus bacteria that is resistant to certain antibiotics calledbeta-lactams. These antibiotics include methicillin and other morecommon antibiotics such as oxacillin, penicillin, and amoxicillin.

Mycobacteria are non-motile, pleomorphic rods, related to theActinomyces. They are relatively impermeable to various basic dyes butonce stained, they retain the dyes with tenacity. They resistdecolorization with acidified organic solvents and are therefore calledacid-fast. They may appear to be Gram positive, but they take up thestain weakly and irregularly and without requiring iodine treatment toretain it.

On the basis of growth rate, catalase and niacin production, andpigmentation in light or dark, mycobacteria are classified into membersof the Mycobacterium tuberculosis complex (including, but not limitedto, M tuberculosis, M bovis, Mycobacterium bovis BCG, Mycobacteriumafricanum, Mycobacterium microti, Mycobacterium canettii, Mycobacteriumcaprae, and Mycobacterium pinnipedii) and nontuberculous species. MostMycobacteria are found in habitats such as water or soil. However, a feware intracellular pathogens of animals and humans. Mycobacteriumtuberculosis, along with M. bovis, M. africanum, and M. microti, allcause the disease known as tuberculosis (TB). Each member of the TBcomplex is pathogenic, but M. tuberculosis is pathogenic for humanswhile M. bovis is usually pathogenic for animals. Tuberculousmycobacteria enter the alveoli by airborne transmission. They resistdestruction by alveolar macrophages and multiply, forming the primarylesion or tubercle; they then spread to regional lymph nodes, entercirculation, and reseed the lungs. Tissue destruction results fromcell-mediated hypersensitivity.

Mycobacterium abscessus is part of the Mycobacterium fortuitum complex,a group of rapidly-growing Mycobacteria that are ubiquitous in theenvironment (soil and water). Mycobacterium fortuitum complex, forexample, includes: Mycobacterium fortuitum, Mycobacterium peregrinum,Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacteriummucogenicum. Mycobacterium fortuitum complex isolates are responsiblefor almost all human infections caused by rapidly-growing Mycobacteria.Infections range from localized wound infections to respiratory diseaseto serious disseminated infections. Mycobacterium abscessus usuallycauses respiratory and soft tissue/skin infections.

Mycobacterium avium complex (MAC) is a commonly isolated group ofMycobacteria and is widely distributed in nature (water, soils, birdsand other animals and dust). The M. avium complex includes M. avium andM. intracellulare. Infections in immunocompetent patients are usuallypulmonary; infections in immunosuppressed patients (e.g., patients whocontracted AIDS or patients whose CD4+ cell counts are less than 200)are usually disseminated. MAC isolates are often drug resistant anddifficult to treat; treatment is only recommended for theimmunosuppressed and in cases of repeated isolation where clinicalsymptoms exist.

Mycobacterium chelonae is part of the Mycobacterium fortuitum complex, agroup of rapidly-growing Mycobacteria that are ubiquitous in theenvironment (soil and water). Mycobacterium chelonae usually causes softtissue and skin infections. It is necessary to differentiate theMycobacterium fortuitum group (Mycobacterium fortuitum and Mycobacteriumperegrinum) from Mycobacterium chelonae and Mycobacterium abscessus asthe latter two species of the complex are very resistant toanti-mycobacterial drug therapies.

Mycobacterium fortuitum and Mycobacterium peregrinum are part of theMycobacterium fortuitum complex. Mycobacterium fortuitum complexincludes: Mycobacterium fortuitum, Mycobacterium peregrinum,Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacteriummucogenicum. Mycobacterium fortuitum complex isolates are responsiblefor almost all human infections caused by rapidly-growing Mycobacteria.The spectrum of diseases caused by these organisms includes soft-tissueabscesses, sternal wound infections after cardiac surgery, prostheticvalve endocarditis, disseminated and localized infection inhaemodialysis and peritoneal dialysis patients, pulmonary disease,traumatic wound infection, and disseminated disease often with cutaneouslesions.

Mycobacterium kansasii is a significant cause of chronic pulmonarydisease in humans, which resembles the disease caused by Mycobacteriumtuberculosis. In contrast to Mycobacterium tuberculosis infection,however, there is minimal risk of human to human transmission. Riskfactors often predispose infection (underlying pulmonary disease,cancer, alcoholism, and the like). Mycobacterium kansasii is commonlyisolated from water samples; infection is thought to be via aerosolroute. Mycobacterium kansasii is the second most common cause ofmycobacterial infection in AIDS patients (after Mycobacterium aviumcomplex).

Mycobacterium marinum is a fish pathogen found in environmental waters.It grows best below 33° C. and causes a tuberculosis-like disease infish and a chronic skin lesion known as “swimming pool granuloma” inhumans. Infection is acquired by injury of a limb around a home aquariumor marine environment and can lead to a series of ascending subcutaneousabscesses. M. marinum resembles M. kansasii in being photochromogenic.M. marinum varies in susceptibility to antimicrobial agents.

Previously termed “Mycobacterium chelonae-like organisms (MCLO)”Mycobacterium mucogenicum is part of the Mycobacterium fortuitumcomplex. Mycobacterium mucogenicum is the rare cause of human disease;it is estimated that most respiratory isolates are non-pathogenic(except in immunocompromised patients) while most non-respiratoryisolates are pathogenic (usually wound infections). Mycobacteriummucogenicum isolates have a mucoid appearance on laboratory media.

Mycobacterium scrofulaceum is a common cause of lymphadenitis inchildren aged 1 to 3 years. Lymphadenitis usually involves a single nodeor a cluster of nodes in the submandibular area. Characteristically, thenodes enlarge slowly over a period of weeks. There are very few local orsystemic symptoms. Untreated, the infection will usually point to thesurface, rupture, form a draining sinus and eventually calcify.Infection in other tissues occurs occasionally. A very few casesresembling progressive primary tuberculosis have been encountered inchildren. In children, metastatic bone disease may be prominent.Colonies are usually yellow-orange even when grown in the dark(scotochromogenic). They are usually resistant to antituberculosis drugsin vitro.

Mycobacterium ulcerans, found mainly in Africa and Australia, grow onlybelow 33° C. It causes chronic deep cutaneous ulcers in man and usuallyproduces lesions in the cooler parts of the body. Mycobacterium ulceranshas a unique drug sensitivity pattern, i.e., resistance to INH andethambutol and susceptibility to streptomycin and rifampin. Humandisease responds poorly to drug treatment and extensive excisionfollowed by skin grafting is often necessary.

Studies have suggested that the source of human Mycobacterium xenopiinfection is aerosolized water, similar to Mycobacterium avium complex.Mycobacterium xenopi is not as ubiquitous in all water supplies asMycobacterium avium, preferring to grow in hot water supplies. Forimmunocompetent patients, pulmonary disease is most common; risk factorsinclude underlying lung disease, heavy smoking, alcohol abuse andexposure to water containing these Mycobacteria. AIDS is another riskfactor for Mycobacterium xenopi infection, often leading to disseminatedinfection in these patients.

The subject or patient in need of treatment, in one embodiment, has apulmonary disease. In one embodiment, the pulmonary disease is cysticfibrosis, bronchiectasis, pneumonia, or chronic obstructive pulmonarydisease (COPD).

In another embodiment, the subject or patient in need of treatment hasosteomyelitis. In another embodiment, the subject or patient in need oftreatment has endocarditis, bronchitis, hepatitis, myocarditis, and/ornephritis. In another embodiment, the subject or patient in need oftreatment has bacteremia. In another embodiment, the subject or patientin need of treatment has a skin or connective tissue infection,including, but not limited to, folliculitis, cellulitis, furuncules, orpymyositis. In another embodiment, the subject or patient in need oftreatment has a wound or surgical site infection.

The therapeutic agents in the compositions of the present invention aredelivered in therapeutically effective amounts. Combined with theteachings provided herein, by choosing among the various activecompounds and weighing factors such as potency, relativebioavailability, patient body weight, severity of adverse side-effectsand mode of administration, an effective prophylactic or therapeutictreatment regimen may be planned which does not cause substantialtoxicity and yet is effective to treat the particular subject. Theeffective amount for any particular application may vary depending onsuch factors as the disease or condition being treated, the particulartherapeutic agent(s) being administered, the size of the subject, or theseverity of the disease or condition. One of ordinary skill in the artmay determine empirically the effective amount of a particulartherapeutic agent(s) without necessitating undue experimentation. In oneembodiment, the highest safe dose according to some medical judgment isemployed. The terms “dose” and “dosage” are used interchangeably herein.

For any compound described herein the therapeutically effective amountmay be initially determined from preliminary in vitro studies and/oranimal models. A therapeutically effective dose also may be determinedfrom human data for therapeutic agent(s), which have been tested inhumans and for compounds, which are known to exhibit similarpharmacological activities, such as other related active agents. Theapplied dose may be adjusted based on the relative bioavailability andpotency of the administered compound. Adjusting the dose to achievemaximal efficacy based on the methods described above and other methodsis well within the capabilities of the ordinarily skilled artisan.

According to some embodiments, the stabilized lipid-based glycopeptideantibiotic is administered in an amount greater than a minimuminhibitory concentration (MIC) for the pulmonary infection. For example,the MIC of the pulmonary infection is at least about 0.10 micrograms/mL,about 0.10 to 10 micrograms/mL, or about 0.10 to 5 micrograms/mL.

According to another embodiment, following administration of thestabilized lipid-based glycopeptide antibiotics, the Log₁₀ CFU(colony-forming unit) in the lung of the subject, or in sputum or tissuecollected from the lung, are reduced. Log₁₀ CFU is the log value ofcolony-forming unit (CFU). In microbiology, colony-forming unit (CFU orcfu) refers to a measure of viable bacterial numbers. Unlike directmicroscopic counts where all cells, dead and living, are counted, CFUmeasures viable cells. According to some embodiments, followingadministration of the stabilized lipid-based glycopeptide antibiotics,the Log₁₀ CFU in the lung of the subject can be reduced by at leastabout 0.5, about 1.0, about 1.5, about 2.0, or about 2.5. According toanother embodiment, the total CFU in the lung is less than about 1.0,about 0.75, about 0.5, or about 0.25 after administration of thestabilized lipid-based glycopeptide antibiotic formulation. According toanother embodiment, the pulmonary infection in the lung of the subjectis reduced following administration of the stabilized lipid-basedglycopeptide antibiotics. According to another embodiment, the pulmonaryinfection in the lung of the subject is eradicated followingadministration of the stabilized lipid-based glycopeptide antibiotics.According to another embodiment, the pulmonary infection is reduced morethan by inhalation treatment with the same dose of free glycopeptideantibiotic. According to another embodiment, the rate of reduction oreradication of the pulmonary infection in a population of subjects ishigher with treatment with the lipid based glycopeptide antibioticformulation when compared to a population treated with the same dose offree inhaled glycopeptide antibiotic. According to another embodiment,the reduction of infection across a population treated with the inhaledstabilized glycopeptide antibiotic formulation is at least about 20,about 30 , about 40 , about 50, about 70, about 80, or about 90% greaterwhen compared to treatment with inhaled free glycopeptide antibiotic.According to another embodiment, the pulmonary infection is reduced in ashorter period of time when compared to treatment with the same dose ofinhaled free vancomycin. According to another embodiment, the time torecurrence of pulmonary infection or need for medical intervention isextended to a longer period of time when compared to treatment with thesame dose of inhaled free vancomycin.

According to another embodiment, the stabilized lipid-based glycopeptideantibiotic of the described invention can be administered by inhalationas a nebulized spray, powder, or aerosol, or by intratrachealadministration. According to another embodiment, the administration isless frequent and/or has an enhanced therapeutic index compared toinhalation of the free drug or a parenteral form of the drug.Additionally, the time for administering the desired therapeutic dose ofglycopeptide antibiotic is reduced compared to inhalation of the freedrug. Thus, in some embodiments, the lipid-based glycopeptide antibioticformulation is more effective than inhalation of the same amount of thefree drug. Liposomes or other lipid formulations are particularlyadvantageous due to their ability to protect the drug while beingcompatible with the lung lining or lung surfactant. While not beingbound by any particular theory, it is believed that the lipid-basedglycopeptide antibiotic formulation of the present invention has a depoteffect in the lung. As such, the lipid-based glycopeptide antibioticmaintains its therapeutic bioavailability for a period of time afteradministration by inhalation is complete. According to some embodiments,this period of time is longer than the amount of time that freeglycopeptide antibiotic remains therapeutically available. For example,the therapeutic bioavailability of the lipid-based glycopeptideantibiotic may be longer than 3, 4, 5, 6, 7, 8, 9 or 10 days aftertreatment, or even longer than two weeks after administration.

According to some embodiments, at least about 25% of the glycopeptideantibiotic is associated with the liposome after nebulization. Accordingto another embodiment, at least about 50% or at least about 60% of theglycopeptide antibiotic is associated with the liposome afternebulization. According to another embodiment, about 50 to 95%, about 50to 80% or about 60 to 75% of the glycopeptide antibiotic is associatedwith the liposome after nebulization.

According to some embodiments, the stabilized lipid-based glycopeptideantibiotic is administered intravenously, subcutaneously, orally,nasally, intraperitoneally, sublingually, bucally, transdermally, bysubcutaneous or subdermal implantation, or intramuscularly.

According to some embodiments, the stabilized lipid-based glycopeptideantibiotic is administered by injection. According to furtherembodiments, the stabilized lipid-based glycopeptide antibiotic isadministered by intravenous injection. According to another embodiment,the administration is less frequent and/or has an enhanced therapeuticindex compared to injection of the free drug or a parenteral form of thedrug. Additionally, the time for administering the desired therapeuticdose of glycopeptide antibiotic is reduced compared to injection of thefree drug. Thus, in some embodiments, the lipid-based glycopeptideantibiotic formulation is more effective than injection of the sameamount of the free drug. According to another embodiment, the time torecurrence of a bacterial infection or need for medical intervention isextended to a longer period of time when compared to treatment with thesame dose of injected free vancomycin. According to some embodiments,the stabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of about 50 to 1000 mg/day, about 100 to 500mg/day, or about 250 to 500 mg/day. According to another embodiment, thedose is about 100 mg/day. According to another embodiment, the dose isabout 200 mg, about 300 mg, about 400 mg, or about 500 mg per day.

According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition is administered at a dose of from about 0.000001mg/kg body weight to about 10 g/kg body weight. According to anotherembodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of from about 0.000002 mg/kg bodyweight to about 10 g/kg body weight. According to another embodiment,the stabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of from about 0.000003 mg/kg body weight to about10 g/kg body weight. According to another embodiment, the stabilizedlipid-based glycopeptide antibiotic composition is administered at adose of from about 0.000004 mg/kg body weight to about 10 g/kg bodyweight. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition is administered at a dose of fromabout 0.000005 mg/kg body weight to about 10 g/kg body weight. Accordingto another embodiment, the stabilized lipid-based glycopeptideantibiotic composition is administered at a dose of from about 0.000006mg/kg body weight to about 10 g/kg body weight. According to anotherembodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of from about 0.000007 mg/kg bodyweight to about 10 g/kg body weight. According to another embodiment,the stabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of from about 0.000008 mg/kg body weight to about10 g/kg body weight. According to another embodiment, the stabilizedlipid-based glycopeptide antibiotic composition is administered at adose of from about 0.000009 mg/kg body weight to about 10 g/kg bodyweight. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition is administered at a dose of fromabout 0.00001 mg/kg body weight to about 10 g/kg body weight. Accordingto another embodiment, the stabilized lipid-based glycopeptideantibiotic composition is administered at a dose of from about 0.00002mg/kg body weight to about 10 g/kg body weight. According to anotherembodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of from about 0.0003 mg/kg bodyweight to about 10 g/kg body weight. According to another embodiment,the stabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of from about 0.00004 mg/kg body weight to about10 g/kg body weight. According to another embodiment, the stabilizedlipid-based glycopeptide antibiotic composition is administered at adose of from about 0.00005 mg/kg body weight to about 10 g/kg bodyweight. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition is administered at a dose of fromabout 0.00006 mg/kg body weight to about 10 g/kg body weight. Accordingto another embodiment, the stabilized lipid-based glycopeptideantibiotic composition is administered at a dose of from about 0.00007mg/kg body weight to about 10 g/kg body weight. According to anotherembodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of from about 0.00008 mg/kg bodyweight to about 10 g/kg body weight. According to another embodiment,the stabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of from about 0.00009 mg/kg body weight to about10 g/kg body weight. According to another embodiment, the stabilizedlipid-based glycopeptide antibiotic composition is administered at adose of from about 0.0001 mg/kg body weight to about 10 g/kg bodyweight. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition is administered at a dose of about0.0005 mg/kg body weight. According to another embodiment, thestabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of about 0.001 mg/kg body weight. According toanother embodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of about 0.005 mg/kg body weight.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition is administered at a dose of about 0.01 mg/kgbody weight. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition is administered at a dose of about0.1 mg/kg body weight. According to another embodiment, the stabilizedlipid-based glycopeptide antibiotic composition is administered at adose of about 1 mg/kg body weight. According to another embodiment, thestabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of about 10 mg/kg body weight. According toanother embodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of about 20 mg/kg body weight.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition is administered at a dose of about 30 mg/kg bodyweight. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition is administered at a dose of about40 mg/kg body weight. According to another embodiment, the stabilizedlipid-based glycopeptide antibiotic composition is administered at adose of about 50 mg/kg body weight. According to another embodiment, thestabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of about 60 mg/kg body weight. According toanother embodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of about 70 mg/kg body weight.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition is administered at a dose of about 80 mg/kg bodyweight. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition is administered at a dose of about90 mg/kg body weight. According to another embodiment, the stabilizedlipid-based glycopeptide antibiotic composition is administered at adose of about 100 mg/kg body weight. According to another embodiment,the stabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of about 110 mg/kg body weight. According toanother embodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of about 120 mg/kg body weight.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition is administered at a dose of about 130 mg/kg bodyweight. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition is administered at a dose of about140 mg/kg body weight. According to another embodiment, the stabilizedlipid-based glycopeptide antibiotic composition is administered at adose of about 150 mg/kg body weight. According to another embodiment,the stabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of about 160 mg/kg body weight. According toanother embodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of about 170 mg/kg body weight.According to another embodiment, the stabilized lipid-based glycopeptideantibiotic composition is administered at a dose of about 180 mg/kg bodyweight. According to another embodiment, the stabilized lipid-basedglycopeptide antibiotic composition is administered at a dose of about190 mg/kg body weight. According to another embodiment, the stabilizedlipid-based glycopeptide antibiotic composition is administered at adose of about 200 mg/kg body weight. According to another embodiment,the stabilized lipid-based glycopeptide antibiotic composition isadministered at a dose of about 250 mg/kg body weight. According toanother embodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered at a dose of about 500 mg/kg body weight.

According to some embodiments, the stabilized lipid-based glycopeptideantibiotic composition is administered intratracheally. According toanother embodiment, the stabilized lipid-based glycopeptide antibioticcomposition is administered via inhalation. According to someembodiments, the stabilized lipid-based glycopeptide antibioticcomposition is administered via a nebulizer. According to anotherembodiment, administering occurs parenterally. According anotherembodiment, administering occurs intravenously. According to anotherembodiment, administering occurs intramuscularly. According to anotherembodiment, administering occurs intraperitoneally.

According to another embodiment, the composition is administered 1 timeto 4 times a day. According to another embodiment, the composition isadministered once a day, twice a day, three times a day or four times aday. According to another embodiment, the composition is administered ina daily treatment cycle for a period of time, or is administered in acycle of every other day, every third day, every fourth day, every fifthday, every 6^(th) day or once a week for a period of time, the period oftime being from one week to several months, for example, 1, 2, 3, or 4weeks, or 1, 2, 3, 4, 5, or 6 months.

The described invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Unless indicated otherwise, parts are parts byweight, molecular weight is weight average molecular weight, temperatureis in degrees Centigrade, and pressure is at or near atmospheric.

Example 1 Vancomycin Stability in Aqueous Solution Reagents andEquipment

Cholesterol (Cat. No. 700000P) and dipalmitoylphosphatidylcholine (DPPC,Cat. No. 850355P) were purchased from Avanti Polar Lipids CertifiedReference Standard. Vancomycin Chloride (Lot HVN0701303) was purchasedfrom North China Pharmaceutical Group. Methanol (HPLC grade, Cat No.AH230-4), Acetonitrile (HPLC grade), Ammonium Hydroxide (HPLC grade)were purchased from Burdick & Jackson. Glacial Acetic Acid (HPLC grade;JT9515-3) and Sodium Chloride (ACS Reagent; Cat. No. 3628-05) werepurchased from JT Baker. Purified water was prepared by using Milli-Q18.0 MΩ water.

High Performance Liquid Chromatography (HPLC) is from Shimadzu (10 AVPequipped UV-detector and CORONA Charged Aerosol Detector (CAD) and HPLCsoftware is from LC Solutions™. Refrigerated multipurpose centrifuge(5810R) is from Eppendorf (Westbury, N.Y.).

Vancomycin Normal Phase HILIC HPLC Assay

A vancomycin Normal Phase HILIC HPLC assay was performed using aShimadzu 10AVP HPLC system equipped with UV detector (CAD optional)under the following conditions:

-   -   Column: ZIC-HILIC 150×4.6 mm, 3.5 um, 200 A (SeQuant)    -   Column temperature: 30 C    -   Mobile phase: Acetonitrile 37%, Methanol 24%, water 39%, Acetic        acid 0.08%, Ammonium hydroxide 0.02%    -   Flow rate: 0.5 mL/min, Isocratic    -   Injection volume: 20 μL    -   Samples are dissolved in mobile phase. A mix of mobile phase        with n-propanol 4:1 can be used when preparing lipid containing        samples.    -   Vancomycin standards: useful range 10-200 mg/mL, dissolved in        mobile phase. Calibration curve is fitted by a linear function    -   UV detector: Wavelength 280 nm    -   Chromatogram: retention time for        -   CDP-I-M: about 14 min        -   CDP-I-m: about 16 min        -   Vancomycin: about 24 min        -   additional major peaks are at: 20, 22, 27 min. Total            recording time 35 min

Vancomycin Reverse Phase HPLC Assay

A vancomycin Reverse phase HPLC assay (which cannot be used for lipidcontaining samples) was performed using a Shimadzu 10AVP HPLC systemequipped with UV detector (CAD optional) under the following conditions:

-   -   Column: Atlantis® dC18, 5 μ, 250×4.6 mm (Waters)    -   Mobile phase A: acetonitrile 5%, ammonium acetate 15 mM, pH 6.0.        Mobile phase B: acetonitrile 20%, ammonium acetate 15 mM, pH        6.0. Ammonium acetate 1 M solution pH 6.0: approximately 6%        glacial acetic acid (1 M) and 9% ammonium hydroxide 30%        solution.    -   Flow rate: 1 mL/min, Binary gradient

Time (minutes) Solvent B (%) 0 15 15 20 25 35 35 60 40 100 45 100 50 1555 15

-   -   Injection volume: 20 μL    -   Samples are dissolved in mobile phase.    -   Vancomycin standards: useful range 10-200 mg/mL, dissolved in        mobile phase. Calibration curve is fitted by a linear function    -   UV detector: Wavelength 280 nm    -   Chromatogram: retention time for        -   CDP-I-M: about 7 min        -   CDP-I-m: about 16 min        -   Vancomycin: about 26 min        -   Total recording time 55 min

Vancomycin Assay by UV-Vis

The samples to be analyzed were dissolved in n-propanol/H₂O solution(60/40, vol/vol) so that the final vancomycin HC1 concentrations werebetween 0 μg/ml and 200 μg/ml. Pre-dilution with H₂O may be necessary ifthe vancomycin concentration in the original sample is >10 mg/mL. Theconcentrations of the samples were calculated by comparing theirabsorbance at 280 nm with that of a linear standard curve (standardswere prepared from raw material at 0, 50, 100, 150 and 200 μg/ml).

Alternatively, samples were dissolved in the mobile phase used for theHILIC HPLC method mixed with n-propanol at 3:1 vol. In the latter case,the vancomycin extinction coefficient was 5.4 mg/mL⁻¹cm⁻¹, or about 7800M⁻¹cm⁻¹ (1 A per 185 μg/ml)

Lipid HPLC Assay

A lipid HPLC assay was performed using a Shimadzu 10AVP HPLC systemequipped with a Corona Charge Aerosol Detector (CAD) under the followingconditions and parameters:

-   -   Column: Phenomenex Luna 3 μ, C8(2), 75×4.6 mm    -   Column temperature: 30° C.    -   Mobile phase: Acetonitrile 43%, n-Propanol 43%, water 14%,        Acetic acid 0.1%, TEA 0.1%    -   Flow rate: 1 mL/min, Isocratic    -   Injection volume: 20 μL    -   Samples were dissolved in Solution A: Acetonitrile 30%,        n-Propanol 30%, water 40%    -   Lipid standards (DPPC/Cholesterol 20/10, 30/15, and 40/20 μg/mL)    -   Calibration is fitted by a linear function    -   Chromatogram: retention time for cholesterol ˜4 min, DPPC ˜6        min, total recording time 10 min

Samples of Vancomycin Solution for Stability Study

Vancomycin solution samples (1 mL of 200 mg/mL) were put in 4 mL glassvials and incubated at the indicated temperature. Selected samples werealso incubated at a lower concentration (20 mg/mL).

Normal Phase HILIC HPLC assay for Vancomycin

Previously, a reverse phase HPLC assay was developed and used in thefirst study on development of liposomal vancomycin (U.S. Publication No.US 2009-0104257, entitled “Liposomal Vancomycin Formulations,” which isincorporated herein by reference in its entirety). The method employedan Atlantis® dC18 column (Waters) and used a binary mobile phasegradient with Acetonitrile concentration changing 7% to 20% in water.The mobile phase, however, was too polar to dissolve lipid containingsamples. Organic phase extraction was tried but had challenges.

In the present invention, a new assay, i.e., Normal phase HILIC(Hydrophilic Interaction Liquid Chromatography) HPLC assay wasdeveloped. The new assay utilizes a ZIC-HILIC column (SeQuant) andseparates compounds by polar differences. Moreover, the HILIC method canuse less polar mobile phase and thus is able to dissolve both vancomycinand lipid (see Materials and Methods section). Particularly, the mobilephase was selected as: Acetonitrile 37%, Methanol 24%, water 39%, Aceticacid 0.08%, Ammonium hydroxide 0.02%. The drawbacks of this method are:high (but manageable) sensitivity to mobile phase composition, and widerpeaks compared to RP assay. See FIG. 4 for a typical chromatograph.

Chemical Stability of Vancomycin in Aqueous Solution

Samples of vancomycin solutions at a concentration of 200 mg/mL and pH5.0, 5.5, 6.0, and 6.5 were prepared by adding the appropriate amount ofNaOH solution (50 mM or 100 mM). In buffered samples, an appropriateamount of buffer or amino acid additive was added. The pH values werechosen to cover the range between the lowest and highest acceptablevalues of pH, including pH 5.0 for lipid stability reasons and pH 6.5,above which degradation is expected to be impractically fast.

The concentration of 200 mg/mL was chosen to mimic what a highintra-liposomal might be but was no higher than this for practicalhandling purposes. All samples were liquid solutions upon preparation.During incubation, most samples turned into a gel-like consistency andlater solidified further into a wax-like form. In some samples with ahigh level of degradation, crystal granules were observed embedded inthe formed gel.

The Experiment was started with vancomycin samples, in which pH wasadjusted by adding NaOH or an organic base (e.g., Tris-base, EthanolAmine (EOA), or Tri-Ethanol Amine (TEOA)) as shown in Table 7. Forcomparison, lower concentration solutions (20 mg/mL) also were preparedby diluting 200 mg/mL samples 10-fold in water. Samples were incubatedat 4° C. (in refrigerator) and at ambient temperature (about 23° C.,which is referred herein to as “room temperature” or “RT”).

TABLE 7 Samples with pH adjusted by NaOH or organic bases Base addedBase Used pH mol/mol NaOH 5.0 0.16 5.5 0.18 6.0 0.20 6.5 0.28 TRIS 6.00.22 6.5 0.31 TEOA 6.0 0.23 EOA 6.5 0.30 TEOA (Tri-Ethanol amine), EOA(Ethanol amine)

TABLE 8 Degradation of vancomycin: Effect of pH, presence of organicbuffers, ethanol, and ammonium ions, Temperature = 4° C. (CDPI-m +CDPI-M) % at week: Slope 0 1 2 4 8 12 %/w R{circumflex over ( )}2 Sample200 mg/mL NaOH pH 5 0.5 0.68 0.74 0.93 1.40 1.54 0.09 0.97 NaOH pH 550.5 0.73 1.07 1.11 1.67 2.39 0.15 0.98 NaOH pH 6 0.5 0.74 1.38 1.69 2.602.90 0.20 0.94 NaOH pH 65 0.5 1.07 1.66 2.55 5.78 4.46 0.39 0.78 NaOH pH5 + NH4Cl 0.5 0.77 0.80 0.92 1.60 1.77 0.11 0.96 NaOH pH 6 0.5 0.74 1.381.69 0.31 0.92 NaOH pH 6 + EtOH 0.5 0.67 1.19 2.28 0.46 0.97 Tris pH 60.5 0.69 1.47 1.82 0.35 0.91 TEOA pH 6 0.5 0.79 1.04 2.11 0.40 0.97 EOApH 6 0.5 0.79 1.22 2.19 0.43 0.99 NaOH pH 6.5 0.5 1.07 1.66 2.55 0.510.99 NaOH pH 6.5 + EtOH 0.5 0.93 2.06 2.15 0.43 0.81 Tris pH 6.5 0.51.32 2.20 2.78 0.56 0.93 TEOA pH 6.5 0.5 1.15 1.78 2.87 0.59 1.00 EOA pH6.5 0.5 0.98 1.94 3.08 0.66 0.99 Sample 20 mg/mL NaOH pH 5 0.5 0.90 1.241.69 3.29 4.52 0.34 1.00 NaOH pH 55 0.5 1.23 1.34 2.29 3.59 4.80 0.350.99 NaOH pH 6 0.5 1.34 1.61 2.09 3.09 3.31 0.22 0.90 NaOH pH 65 0.51.11 2.05 2.97 2.16 2.42 0.12 0.38

A higher concentration and lower pH, vancomycin was more stable (Table8, FIG. 5). At low concentration, the effect seemed to be unclear oreven reversed. The main difference in low and high concentration sampleswas their physical state. At high concentration vancomycin over timeformed a gel-like structure. At low concentration, it stayed liquiduntil a white precipitate started to form. Excluding sodium ions andusing instead organic buffer bases to adjust pH did not changedegradation kinetics noticeably (FIG. 6).

Overall, the summary plot in FIG. 7 shows that at high concentration andlow pH vancomycin can be somewhat stable with a degradation rate ofabout 0.1% per week. This, however, is not believed to be good enough todevelop a practical commercial product. Addition of an equimolar amountof ammonium ions (NH₄Cl) did not improve vancomycin stability. Ethanolat 10% efficiently prevented vancomycin gelation and precipitation, butagain did not slow down its degradation rate.

At room temperature, degradation rates were about ten-fold higher butthe data showed greater variability (Table 9 and FIGS. 8-10).

TABLE 9 The Effect of pH, presence of organic buffers, ethanol, andammonium ions on degradation of vancomycin at room temperature (RT)(CDPI-m + CDPI-M) % at week: Slope 0 1 2 4 8 12 %/w R{circumflex over( )}2 Sample 200 mg/mL NaOH pH 5 0.5 1.34 2.47 6.28 10.37 17.3 1.39 0.99NaOH pH 55 0.5 1.86 3.30 5.97 11.0 16.2 1.30 1.00 NaOH pH 6 0.5 2.043.23 6.66 14.0 1.69 1.00 NaOH pH 65 0.5 3.16 5.36 11.1 11.8 1.42 0.83NaOH pH 5 + NH4Cl 0.5 1.49 1.99 3.63 9.4 1.10 0.97 NaOH pH 6 0.5 2.043.23 6.66 1.53 1.00 NaOH pH 6 + EtOH 0.5 2.27 3.65 5.86 1.32 0.99 TrispH 6 0.5 2.25 4.23 11.9 2.88 0.96 TEOA pH 6 0.5 2.85 5.12 7.36 1.70 0.96EOA pH 6 0.5 2.45 5.79 7.38 1.75 0.92 NaOH pH 6.5 0.5 3.16 9.65 11.12.77 0.86 NaOH pH 6.5 + EtOH 0.5 2.92 4.51 7.87 1.80 0.99 Tris pH 6.50.5 3.32 5.05 8.16 1.86 0.98 TEOA pH 6.5 0.5 3.38 7.97 3.73 0.98 EOA pH6.5 0.5 3.70 8.25 9.79 2.34 0.88 Sample Temp RT NaOH pH 5 0.5 2.12 2.876.03 10.8 19.5 1.53 0.98 NaOH pH 55 0.5 2.22 4.44 5.15 5.08 13.6 0.890.84 NaOH pH 6 0.5 2.80 4.89 7.97 9.85 1.13 0.89 NaOH pH 65 0.5 4.169.65 10.8 11.3 1.20 0.64

There was no clear effect of buffers, ammonium ions, or added ethanol.The high variability in data might be explained by the non-homogeneousnature of samples after long incubation at room temperature. Samplessolidified and exhibited a number of crystalline granules embeddedwithin. Taking an aliquot of sample for analysis often resulted ingetting a portion of sample of varying consistency.

Example 2 Effect of Amino Acid Additives on Chemical Stability ofVancomycin

It has been known that some peptides as short as two or three aminoacids can bind tightly to vancomycin and thus stabilize its structureagainst deamidation (Harris et al., 1985, J. Antibiot, 38(1):51-7). Twosuch peptides have been identified, i.e., Ac-D-Ala-D-Ala andDi-Ac-L-Lys-D-Ala-D-Ala. While they potentially could be used to provideimproved vancomycin stability, they are not cost-effective means ofimproving vancomycin stability.

Below is a list of amino acids identified for testing (Table 10). Aminoacids were added on a mole per mole base unless otherwise stated. FinalpH was adjusted to pH 5.0 or pH 5.5 where indicated by adding anappropriate amount of NaOH. Samples at a concentration 200 mg/mL wereplaced 1 mL each into 4 mL glass vials and incubated at a certaincontrolled temperature. Samples were analyzed by HILIC HPLC, and CDP %was calculated as a sum of CDP-I-m and CDP-I-M % to total vancomycin andits products.

TABLE 10 Amino Acids And Derivatives Used In Stability Study. Amino acidChemical structure ALA-ALA

GLY, Glycine

ALA, Alanine 2-Aminopropionic acid

bALA, beta-Alanine 3-Aminopropionic acid

3-ABA 3-Aminobutanoic acid

GABA 4-Aminobutanoic acid

GLU, Glutamic acid 2-Aminopentanedioic acid

ASP, Aspartic acid 2-Aminobutanedioic acid

Sarcosine N-Methylglycine

IDAA Iminodiacetic acid

GLY-GLY Glycyl-glycine

Bicine

Tricine

A detailed stability data table at 4° C. is presented in Table 11 andFIG. 11. A significant difference was found in the rate of degradation,with the worst rate found for samples incubated with L-ALA, b-ALA,3-ABA, and GLY. At the same time, great improvement was seen when usingBicine, Imino-diacetic acid (IDAA), glycylglycine (GLY-GLY), and GLU at2:1 mol ratio with vancomycin. A rate of as low as 0.01% per week wasobserved for some compounds, including Bicine, IDAA, and GLY-GLY. Incomparison, control compounds that do not comprise an amino acid orderivative thereof exhibited a rate of 0.09%. Thus, the stabilizedglycopeptide antibiotic composition comprising an amino acid such as,for example, Bicine, IDAA, or GLY-GLY was at least 88% more stable at 4°C. than a glycopeptide antibiotic that does not comprise an amino acidor derivative thereof.

TABLE 11 Degradation of vancomycin data table: Effect of amino acidadditives at 4° C. Amino acid (CDPI-m + CDPI-M) % at week: Slope added 01 2 4 8 12 %/w NaOH pH 5 0.50 0.61 0.69 0.93 1.39 1.49 0.09 bALA 0.600.81 0.89 1.07 2.09 3.18 0.21 ALA 0.60 0.69 0.76 0.97 1.46 2.34 0.14GABA pH 5.5 0.60 0.91 0.89 1.11 1.08 1.81 0.08 GLY 0.60 0.76 0.77 0.961.68 2.14 0.13 D-ALA 0.60 0.74 0.70 0.92 1.48 1.27 0.07 3-ABA 0.60 0.650.57 0.95 1.57 2.29 0.15 GLUt_NH3 0.60 0.83 0.72 0.84 1.44 2.03 0.12D-GLUt 0.60 0.68 0.75 0.89 1.14 1.43 0.07 ASP 0.60 0.75 0.65 0.82 1.201.09 0.05 D-ASP 0.60 0.74 0.74 0.63 1.03 1.35 0.06 Bicine 0.60 0.66 0.580.61 0.77 0.67 0.01 Tricine 0.60 0.62 0.78 0.69 1.02 1.32 0.06 Sarcosine0.54 0.65 0.70 1.11 1.08 0.07 IDAA 0.50 0.54 0.66 0.74 0.56 0.01 GLY-GLY0.60 0.66 0.50 0.66 0.69 0.01 GLUt2 pH 5 0.60 0.61 0.67 0.86 0.92 0.870.03 GLUt2 pH 5.5 0.60 0.82 1.06 0.79 0.83 1.29 0.04

TABLE 12 Degradation of vancomycin data table: Rate of degradation at 4°C. with amino acid additives relative to control. Rate of degradationAmino acid Structure relative to control D-ALA D-Alanine

0.36% weight/week ASP Aspartic acid

0.42% weight/week Bicine

0.18% weight/week D-GLU D-Glutamic acid

0.37% weight/week GLY-GLY Glycylglycine

0.31% weight/week IDAA Iminodiacetic acid

0.24% weight/week

A similar study conducted for samples at room temperature is presentedin Table 13 and FIG. 12. Some of the amino acids that did not performvery well at 4° C. were much better in stabilizing vancomycin at roomtemperature. GABA, D-ALA, D-GLU, and ASP reduced degradation to about0.2-0.4% per week, compared to about 1.4% per week in control. Thus, thestabilized glycopeptide antibiotic composition comprising amino acid orderivative thereof such as, for example, GABA, D-ALA, D-GLU, or ASP wasabout 71.4% to about 85.7% more stable at RT than a glycopeptideantibiotic that does not comprise an amino acid or derivative thereof.

TABLE 13 Degradation of vancomycin data table: Effect of amino acidadditives at room temperature (RT). (CDPI-m + CDPI-M) % at week: StopeRatio Sample 0 1 2 4 8 12 %/w RT/4 C. NaOH pH 5 0.50 1.18 2.80 6.58 9.8918.10 1.43 16.3 b-ALA 0.50 2.31 3.40 9.04 12.27 25.30 1.95 9.1 ALA 0.501.76 3.35 9.39 9.02 31.48 2.29 16.3 GABA pH 5.5 0.50 2.20 3.18 4.23 4.446.44 0.41 5.0 GLY 0.50 1.56 3.24 6.14 17.19 21.04 1.85 14.0 D-ALA 0.501.35 1.61 1.73 2.49 3.56 0.22 3.3 3-ABA 0.50 1.51 3.03 6.03 9.23 19.281.48 10.1 GLUt_NH3 0.50 1.94 6.20 4.05 7.42 10.22 0.71 6.1 D-GLUt 0.501.57 1.93 1.68 4.17 5.35 0.38 5.6 ASP 0.50 0.80 2.22 2.27 3.24 4.81 0.337.0 D-ASP 0.50 1.46 2.47 3.80 5.97 7.08 0.54 9.3 Bicine 0.50 0.89 1.473.21 6.76 10.96 0.89 99.4 Tricine 0.50 1.15 2.09 6.21 9.66 17.63 1.4123.9 Sarcosine 0.54 1.17 2.33 6.48 11.47 1.44 19.8 IDAA 0.60 2.25 2.765.39 10.07 1.17 145 GLY-GLY 0.66 0.72 1.24 2.80 5.92 0.69 60.3

The ratio of the rate of conversion at room temperature (RT) to the rateat 4° C. is calculated in the last column in Table 13. This ratio variesdramatically, from 3.3 for D-ALA to 145 for IDAA. This could indicatethat different AA affect differently the conversion reaction ofvancomycin to CDP. Without being limited by theory, the rate could havechanged through a change in activation energy, or through change inother factors such as the functional group orientation or theprobability of collision.

One way to study those factors is to determine the rate of conversion atdifferent temperatures and present the data in the form of an Arrheniusplot. An Arrhenius plot displays the logarithm of kinetic constants(ln(k), ordinate axis) plotted against inverse temperature (1/T,abscissa). For a single rate-limited thermally activated process, anArrhenius plot gives a straight line from which the activation energyand the pre-exponential factor can both be determined.

The Arrhenius equation can be written as:

${\ln (k)} = {{\ln (A)} - {\frac{E_{a}}{R}\; \left( \frac{1}{T} \right)}}$

-   -   where:    -   k=Rate constant    -   A=Pre-exponential factor    -   E_(a)=Activation energy    -   R=Gas constant    -   T=Absolute temperature, K

When plotted in the manner described above, the value of the“y-intercept” will correspond to ln(A), and the gradient of the linewill be equal to −E_(a)/R. The pre-exponential factor, A, is a constantof proportionality that takes into account a number of factors, such asthe frequency of collision between and the orientation of the reactingparticles.

TABLE 14 Degradation of vancomycin at number of different controlledtemperatures. Temp 50 40 30 23 15 4 Slope E R² 10³/T 3.10 3.19 3.30 3.383.47 3.61 K kJ/mol Sample CDP-I rate %/week V_NaOH 42.44 13.53 3.48 1.670.81 0.15 V_Bicine2 28.88 4.91 1.01 0.40 0.14 0.01 V_GLYGLY1 33.90 6.002.03 1.14 0.22 0.05 V_GLYGLY1.5 25.78 3.69 1.57 0.87 0.23 0.04 V_GLYGLY233.12 6.19 1.70 0.97 0.25 0.04 V_GLU2 38.46 5.18 1.50 0.95 0.33 0.05 Ln(CDP-I rate) V_NaOH 3.75 2.60 1.25 0.51 −0.21 −1.92 −10.8 89.7 0.99V_Bicine2 3.36 1.59 0.01 −0.92 −2.00 −4.57 −14.8 123 0.99 V_GLYGLY1 3.521.79 0.71 0.13 −1.52 −3.06 −12.5 104 0.99 V_GLYGLY1.5 3.25 1.31 0.45−0.14 −1.46 −3.23 −11.9 99 0.98 V_GLYGLY2 3.50 1.82 0.53 −0.03 −1.39−3.32 −12.8 106 0.99 V_GLU2 3.65 1.64 0.40 −0.06 −1.11 −3.07 −12.2 1020.98

For this evaluation vancomycin samples at 200 mg/mL and pH adjusted topH 5.5 were used. From the slope of the fitted line on Arrhenius plot(FIG. 13), the activation energy for vancomycin to CDP conversion hasbeen determined (Table 14). In a control sample with no amino acid, theactivation energy was 89.7 kJ/ml. All tested amino acids increasedactivation energy, with Bicine leading to an increase of about 123kJ/mol.

Example 3 Stability of Liposomal Vancomycin-Amino Acid Formulations

Three formulations (see Table 15) of liposomal vancomycin were preparedusing the 3-stream infusion process shown in FIG. 14.

TABLE 15 Composition of liposomal vancomycin batches used for stabilitystudy. Batch AA Lipid Composition Washing NaCl L-VGLUt0330 GLU DPPC/Chol0.90% (50:50 mol %) L-VDGLUt0405 D-GLU DPPC/DPPG/Chol 1.50% (47.5:5:47.5mol %) LPG-VGLUto408 GLU DPPC/Chol 1.50% (50:50 mol %)

Liposomes were washed and concentrated to about 20 mg/mL lipid. Thedrug:lipid ratio was about 0.2. Approximately 1 mL each sample wasincubated at 4° C. and at RT. Over time liposomes settled in the firsttwo batches. Supernatant in those samples were analyzed for vancomycinas a measure of free vancomycin. Degradation of total vancomycin wasmeasured after vortexing the sample and taking an aliquot of 10 μL.

The data are presented in Table 16 and FIGS. 15 and 16. From thislimited incubation time one can see that vancomycin stability insideliposomes is comparable or better than in a solution of vancomycin withGLU added. For example, the rate for vancomycin—GLU (1:1) was 0.12% perweek (Table 11), compared to 0.025 to 0.035% per week (Table 16) whenencapsulated in liposomes.

In addition, an estimate of free vancomycin based on the vancomycinlevel in supernatant indicates modest or low leak at 4° C. and at RT(FIGS. 17 and 18, respectively). Thus, the stabilized lipid-basedglycopeptide antibiotic compositions of the present invention areunexpectedly more stable in comparison to glycopeptide antibioticcompositions that are not lipid-based and/or do not comprise an aminoacid or derivative thereof.

TABLE 16 Degradation rates of vancomycin-GLU inside liposomesDegradation rate, %/w Batch 4° C. RT L-VGLU0330 0.035 0.76 L-VDGLU04050.028 0.47 LPG-VGLU0408 0.025 0.89

In summary, the data indicate that the addition of amino acids and theirderivatives remarkably affected the degradation rate of vancomycin. Themost promising amino acids were Bicine, Imino-diacetic acid (IDAA),glycylglycine (GLY-GLY), and GLU (when used at 2:1 mol ratio withvancomycin). At 4° C., degradation rates as low as 0.01% per week wereobserved. Arrhenius plots for degradation in the presence of key aminoacids could be fitted well to a linear function. The data suggested thatimprovement in stability is caused by an increase in activation energyvalue from 87 kJ/mol to up to 120 kJ/mol. Furthermore, encapsulation inliposomes of the vancomycin-amino acid compositions exhibited superiorchemical stability.

Example 4 Stability of Liposomal Glycopeptide Antibiotic-Amino AcidFormulations

Formulations of liposomal glycopeptide antibiotic comprisingteicoplanin, telavancin, oritavancin, decaplanin or dalbavancin areprepared using the 3-stream infusion process shown in FIG. 14. Lipidcomponents in the formulations comprise DPPC, DPPC+cholesterol, DPPG,DPPG+cholesterol, DPPC+DPPC+cholesterol, or POPC. Amino acid componentsin the formulations comprise Bicine, GLU, GLY-GLY, IDAA, ASP, or D-ALA.

Liposomes are washed and concentrated to about 20 mg/mL lipid.Approximately 1 mL each sample is incubated at 4° C. and at RT. Toassess the stability of the glycopeptide antibiotic in the formulation,supernatant is analyzed for free glycopeptide antibiotic. Degradation oftotal glycopeptide antibiotic is measured after vortexing the sample andtaking an aliquot of 10 μL.

All publications, protocols, patents and patent applications citedherein are incorporated herein by reference in their entireties for allpurposes.

While the described invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the invention. Allsuch modifications are intended to be within the scope of the claimsappended hereto.

1. A stabilized lipid-based glycopeptide antibiotic compositioncomprising: (a) a lipid component; (b) a glycopeptide antibioticcomponent; and (c) an amino acid or a derivative thereof, wherein theamino acid or the derivative thereof stabilizes the glycopeptideantibiotic.
 2. The stabilized lipid-based glycopeptide antibioticcomposition according to claim 1, wherein the amino acid or thederivative thereof reduces the degradation rate of the glycopeptideantibiotic.
 3. The stabilized lipid-based glycopeptide antibioticcomposition according to claim 1, wherein the stabilized glycopeptideantibiotic-amino acid complex is entrapped by the lipid component. 4.The stabilized lipid-based glycopeptide antibiotic composition accordingto claim 1, wherein the antibiotic composition is at least 44% morestable than an antibiotic composition comprising the same lipidcomponent and the same glycopeptide antibiotic component, that does notcomprise an amino acid or a derivative thereof.
 5. The stabilizedlipid-based glycopeptide antibiotic composition according to claim 1,wherein the antibiotic composition is at least 77% more stable than anantibiotic composition comprising the same lipid component and the sameglycopeptide antibiotic component, that does not comprise an amino acidor a derivative thereof.
 6. The stabilized lipid-based glycopeptideantibiotic composition according to claim 1, wherein the antibioticcomposition is at least 88% more stable than an antibiotic compositioncomprising the same lipid component and the same glycopeptide antibioticcomponent, that does not comprise an amino acid or a derivative thereof.7. The stabilized lipid-based glycopeptide antibiotic compositionaccording to claim 1, wherein the composition produces productdegradants at a rate less than 0.05% by weight per week at 4° C.
 8. Thestabilized lipid-based glycopeptide antibiotic composition according toclaim 1, wherein the composition produces product degradants at a rateless than 0.02% by weight per week at 4° C.
 9. The stabilizedlipid-based glycopeptide antibiotic composition according to claim 1,wherein the composition produces product degradants at a rate less than0.01% by weight per week at 4° C.
 10. The stabilized lipid-basedglycopeptide antibiotic composition according to claim 1, wherein thecomposition produces product degradants at a rate less than about 0.4%by weight per week at room temperature.
 11. The stabilized lipid-basedglycopeptide antibiotic composition according to claim 1, wherein thecomposition produces product degradants at a rate less than about 0.2%by weight per week at room temperature.
 12. The stabilized lipid-basedglycopeptide antibiotic composition according to any one of claims 7 to11, wherein the product degradants are crystalline degradation products.13. The stabilized lipid-based glycopeptide antibiotic compositionaccording to claim 1, wherein the lipid component comprises aphospholipid.
 14. The stabilized lipid-based glycopeptide antibioticcomposition according to claim 13, wherein the phospholipid is selectedfrom the group consisting of phosphatidylcholine (PC),phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine(PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), and amixture thereof.
 15. The stabilized lipid-based glycopeptide antibioticcomposition according to claim 1, wherein the lipid component comprisesa sterol.
 16. The stabilized lipid-based glycopeptide antibioticcomposition according to claim 15, wherein the sterol is cholesterol.17. The stabilized lipid-based glycopeptide antibiotic compositionaccording to claim 1, wherein the lipid component comprises aphospholipid and a sterol.
 18. The stabilized lipid-based glycopeptideantibiotic composition according to claim 1, wherein the lipid componentcomprises dipalmitoylphosphatidylcholine (DPPC).
 19. The stabilizedlipid-based glycopeptide antibiotic composition according to claim 1,wherein the lipid component comprises dipalmitoylphosphatidylcholine(DPPC) and cholesterol.
 20. The stabilized lipid-based glycopeptideantibiotic composition according to claim 1, wherein the lipid componentcomprises dipalmitoylphosphatidylglycerol (DPPG).
 21. The stabilizedlipid-based glycopeptide antibiotic composition according to a claim 1,wherein the lipid component comprises dipalmitoylphosphatidylglycerol(DPPG) and cholesterol.
 22. The stabilized lipid-based glycopeptideantibiotic composition according to claim 1, wherein the lipid componentcomprises dipalmitoylphosphatidylcholine (DPPC),dipalmitoylphosphatidylglycerol (DPPG), and cholesterol.
 23. Thestabilized lipid-based glycopeptide antibiotic composition according toclaim 3, wherein the stabilized glycopeptide antibiotic-amino acidcomplex is entrapped in a liposome.
 24. The stabilized lipid-basedglycopeptide antibiotic composition according to claim 23, wherein theliposome has a mean particle size of about 0.05 microns to 10 microns.25. The stabilized lipid-based glycopeptide antibiotic compositionaccording to claim 3, wherein the stabilized glycopeptideantibiotic-amino acid complex is entrapped into a lipid clathrate. 26.The stabilized lipid-based glycopeptide antibiotic composition accordingto claim 3, wherein the stabilized glycopeptide antibiotic-amino acidcomplex is entrapped by a proliposome.
 27. The stabilized lipid-basedglycopeptide antibiotic composition according to any one of claims 1 to26, wherein the glycopeptide antibiotic is vancomycin.
 28. Thestabilized lipid-based glycopeptide antibiotic composition according toany one of claims 1 to 26, wherein the amino acid is D-alanine.
 29. Thestabilized lipid-based glycopeptide antibiotic composition according toany one of claims 1 to 26, wherein the amino acid is aspartic acid. 30.The stabilized lipid-based glycopeptide antibiotic composition accordingto any one of claims 1 to 26, wherein the amino acid derivative isbicine.
 31. The stabilized lipid-based glycopeptide antibioticcomposition according to any one of claims 1 to 26, wherein the aminoacid is D-glutamic acid.
 32. The stabilized lipid-based glycopeptideantibiotic composition according to any one of claims 1 to 26, whereinthe amino acid derivative is glycylglycine (Gly-Gly).
 33. The stabilizedlipid-based glycopeptide antibiotic composition according to any one ofclaims 1 to 26, wherein the amino acid derivative is iminodiacetic acid(IDAA).
 34. The stabilized lipid-based glycopeptide antibioticcomposition according to any one of claims 1 to 26, wherein a molarratio of the glycopeptide antibiotic component to the amino acid oramino acid derivative is from about 1:1 to about 1:4.
 35. The stabilizedlipid-based glycopeptide antibiotic composition according to claim 34,wherein a molar ratio of the glycopeptide antibiotic component to theamino acid or amino acid derivative is from about 1:1 to about 1:2. 36.The stabilized lipid-based glycopeptide antibiotic composition accordingto claim 35, wherein a molar ratio of the glycopeptide antibioticcomponent to the amino acid or amino acid derivative is about 1:1. 37.The stabilized lipid-based glycopeptide antibiotic composition accordingto claim 35, wherein a molar ratio of the glycopeptide antibioticcomponent to the amino acid or amino acid derivative is about 1:2. 38.The stabilized lipid-based glycopeptide antibiotic composition accordingto any one of claims 1 to 26, wherein the composition has a pH of fromabout 5.5 to about 6.5.
 39. The stabilized lipid-based glycopeptideantibiotic composition according to claim 38, wherein the compositionhas a pH of about 5.5.
 40. The stabilized lipid-based glycopeptideantibiotic composition according to any one of claims 1 to 26, whereinconcentration of the glycopeptide antibiotic component in thecomposition is from about 20 mg/mL to about 200 mg/mL.
 41. Thestabilized lipid-based glycopeptide antibiotic composition according toclaim 40, wherein concentration of the glycopeptide antibiotic componentin the composition is about 100 mg/mL.
 42. The stabilized lipid-basedglycopeptide antibiotic composition according to claim 40, whereinconcentration of the glycopeptide antibiotic component in thecomposition is about 200 mg/mL.
 43. The stabilized lipid-basedglycopeptide antibiotic composition according to any one of claims 1 to26, wherein the composition comprises an amino acid component orderivative thereof that is a dipeptide.
 44. The stabilized lipid-basedglycopeptide antibiotic composition according to any one of claims 1 to26, wherein the composition comprises an amino acid component orderivative thereof that is a tripeptide.
 45. The stabilized lipid-basedglycopeptide antibiotic composition according to any one of claims 1 to26, wherein the composition further comprises an excipient.
 46. A methodfor preparing a stabilized lipid-based glycopeptide antibioticcomposition, comprising: infusing, in an in-line fashion, a first streamof a lipid solution containing a lipid component in a solvent with asecond stream of an aqueous solution comprising a glycopeptideantibiotic and an amino acid or a derivative thereof, wherein the aminoacid or the derivative thereof binds to the glycopeptide antibiotic andforms a stabilized glycopeptide antibiotic-amino acid complex, and thestabilized glycopeptide antibiotic-amino acid complex is entrapped bythe lipid component.
 47. The method according to claim 46, wherein thesolvent is ethanol.
 48. The method according to claim 46, wherein thelipid solution comprises about 20 mg/mL of the lipid component.
 49. Themethod according to claim 46, wherein the lipid component comprises aphospholipid.
 50. The method according to claim 49, wherein thephospholipid is selected from the group consisting ofphosphatidylcholine (PC), phosphatidylglycerol (PG),phosphatidylinositol (PI), phosphatidylserine (PS),phosphatidylethanolamine (PE), phosphatidic acid (PA), and a mixturethereof.
 51. The method according to claim 46, wherein the lipidcomponent comprises a sterol.
 52. The method according to claim 51,wherein the sterol is cholesterol.
 53. The method according to claim 46,wherein the lipid component comprises a phospholipid and a sterol. 54.The method according to claim 46, wherein the lipid component comprisesdipalmitoylphosphatidylcholine (DPPC).
 55. The method according to claim46, wherein the lipid component comprises dipalmitoylphosphatidylcholine(DPPC) and cholesterol.
 56. The method according to claim 46, whereinthe lipid component comprises dipalmitoylphosphatidylglycerol (DPPG).57. The method according to claim 46, wherein the lipid componentcomprises dipalmitoylphosphatidylglycerol (DPPG) and cholesterol. 58.The method according to claim 46, wherein the lipid component comprisesdipalmitoylphosphatidylcholine (DPPC) anddipalmitoylphosphatidylglycerol (DPPG).
 59. The method according toclaim 46, wherein the lipid component comprisesdipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol(DPPG), and cholesterol.
 60. The method according to claim 46, whereinthe first stream has a flow rate of about 1 L/min.
 61. The methodaccording to claim 46, wherein the molar ratio of the glycopeptideantibiotic to the amino acid or amino acid derivative is from about 1:1to about 1:4.
 62. The method according to claim 46, wherein the molarratio of the glycopeptide antibiotic to the amino acid or amino acidderivative is from about 1:1 to about 1:2.
 63. The method according toclaim 46, wherein the amino acid is D-alanine.
 64. The method accordingto claim 46, wherein the amino acid is aspartic acid.
 65. The methodaccording to claim 46, wherein the amino acid derivative is bicine. 66.The method according to claim 46, wherein the amino acid is D-glutamicacid.
 67. The method according to claim 46, wherein the amino acidderivative is glycylglycine (Gly-Gly).
 68. The method according to claim46, wherein the amino acid derivative is iminodiacetic acid (IDAA). 69.The method according to claim 46, wherein the aqueous solution comprisesabout 20 mg/mL to about 200 mg/mL of the glycopeptide antibiotic. 70.The method according to claim 46, wherein the aqueous solution comprisesabout 200 mg/mL of glycopeptide antibiotic.
 71. The method according toclaim 46, wherein the aqueous solution comprises about 100 mg/mL ofglycopeptide antibiotic.
 72. The method according to claim 46, whereinthe glycopeptide antibiotic is vancomycin.
 73. The method according toclaim 44, wherein the aqueous solution has a pH from about 5.0 to about6.5.
 74. The method according to claim 46, wherein the aqueous solutionhas a pH of about 5.5.
 75. The method according to claim 46, wherein thesecond stream has a flow rate of about 1.5 L/min.
 76. The methodaccording to claim 46, further comprising infusing a saline solution.77. A method for treating a bacterial infection, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the stabilized lipid-based glycopeptide antibiotic compositionaccording to any one of claims 1-45.
 78. The method according to claim77, wherein the bacterial infection is a pulmonary infection.
 79. Themethod according to claim 77, wherein the subject has bacteremia. 80.The method according to claim 77, wherein the subject has osteomyelitis.81. The method according to claim 77, wherein the glycopeptideantibiotic is vancomycin.
 82. The method according to claim 77, whereinthe bacterial pulmonary infection is caused by Gram-positive bacteria.83. The method according to claim 82, wherein the Gram-positive bacteriacomprise Methicillin-resistant Staphylococcus aureus (MRSA),Streptococcus pneumoniae, Escherichia coli, Klebsiella, Enterobacter,Serratia, Haemophilus, Yersinia pesos, Burkholderia pseudomallei,Burkholderia cepacia, Burkholderia gladioli, Burkholderia multivorans,or Burkholderia vietnamiensis.
 84. The method according to claim 82,wherein the Gram-positive bacteria comprise Mycobacteria.
 85. The methodaccording to claim 84, wherein the Mycobacteria is Mycobacteriumtuberculosis, nontuberculous mycobacterium, Mycobacterium avium complex(MAC), Mycobacterium kansasii, Mycobacterium xenopi, Mycobacteriummarinum, Mycobacterium ulcerans, Mycobacterium fortuitum complex,Mycobacterium abscessus or Mycobacterium xenopi.
 86. The methodaccording to claim 82, wherein the Gram-positive bacteria compriseBurkholderia.
 87. The method according to claim 77, wherein thetherapeutically effective amount is an amount greater than a minimuminhibitory concentration (MIC) for the bacterial pulmonary infection.88. The method according to claim 77, wherein the therapeuticallyeffective amount is from about 50 mg per day to about 1000 mg per day.89. The method according to claim 77, wherein the stabilized lipid-basedglycopeptide antibiotic composition is administered intratracheally. 90.The method according to claim 77, wherein the stabilized lipid-basedglycopeptide antibiotic composition is administered via inhalation. 91.The method according to claim 77, wherein the stabilized lipid-basedglycopeptide antibiotic composition is administered via a nebulizer. 92.The method according to claim 77, wherein the stabilized lipid-basedglycopeptide antibiotic composition is administered 1 to 4 times perday.